Valved cartridge and system

ABSTRACT

This disclosure provides, among other things, a cartridge comprising: (a) a cartridge body comprising a malleable material and having, disposed on a surface of the body, at least one valve body comprising a valve inlet and a valve outlet, each fluidically connected to a fluidic channel; and (b) a layer comprising a deformable material bonded to a surface of the cartridge body and sealing the at least one valve body at points of attachment, thereby forming at least one valve; wherein the at least one valve body is depressed in the cartridge body relative to the points of attachment and wherein the deformable material covering the at least one valve body retains sufficient elasticity after deformation such that in a ground state the valve is open. Also disclosed is an instrument comprising a cartridge interface and a cartridge as described herein engaged with the cartridge interface, wherein (II) the cartridge interface comprises: (A) at least one mechanical actuator, each mechanical actuator positioned to actuate a valve; and (B) at least one motor operatively coupled to actuate a mechanical actuator toward or away from a valve.

REFERENCE TO RELATED APPLICATIONS

This application is a non-provisional application of U.S. Provisionalapplication 62/233,852, filed Sep. 28, 2015 and U.S. Provisionalapplication 62/182,291, filed Jun. 19, 2015, each of which isincorporated herein by reference in its entirety.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

None.

BACKGROUND OF THE INVENTION

Versions of systems including sample cartridges and fluidic systems forsample extraction and analysis are described in, for example, U.S. Pat.Nos. 6,190,616; 6,551,839; 6,870,185; 7,244,961; 8,394,642 and8,431,340; US patent applications 2006/0073484; 2009/0253181;2011/0039303; 2011/0126911; 2012/0181460; 2013/0139895 and 2013/0115607;and International Patent Applications PCT/US2013/130910 andPCT/EP2012/001413.

US patent publication 2003/0197139 refers to a valve for use inmicrofluidic structures.

US patent publication 2009/0314970 refers to a mechanically-actuatedmicrofluidic pinch valve.

US patent publication 2013/0240140 refers to a process for producing amicrofluidic apparatus and related laminating devices.

International publication WO 2012/136333 refers to a heat weldable filmfor labeling plastic polymeric reaction tubes.

U.S. Pat. No. 6,883,774 refers to a microvalve and method of forming amicrovalve.

U.S. Pat. No. 7,318,912 refers to microfluidic systems and methods forcombining discreet fluid volumes.

U.S. Pat. No. 8,313,941 refers to integrated microfluidic controlemploying programmable tactile actuators.

U.S. Pat. No. 8,501,305 refers to a laminate.

The statements in the Background are not necessarily meant to endorsethe characterization in the cited references or admit their availabilityas prior art.

BRIEF SUMMARY OF THE INVENTION

In one aspect disclosed herein is a fluidic device comprising: (a) asubstrate comprising a malleable material and having, disposed on asurface of the body, at least one valve body comprising a valve inletand a valve outlet, each fluidically connected to a fluidic channel; and(b) a layer comprising a deformable material bonded to a surface of thesubstrate and sealing the at least one valve body at points ofattachment, thereby forming at least one valve; wherein the at least onevalve body is depressed in the substrate relative to the points ofattachment and wherein the deformable material covering the at least onevalve body retains sufficient elasticity after deformation such that ina ground state the valve is open. In one embodiment, the fluidic deviceis a microfluidic chip.

In another aspect provided herein is in a cartridge comprising: (a) acartridge body comprising a malleable material and having, disposed on asurface of the body, at least one valve body comprising a valve inletand a valve outlet, each fluidically connected to a fluidic channel; and(b) a layer comprising a deformable material bonded to a surface of thecartridge body and sealing the at least one valve body at points ofattachment, thereby forming at least one valve; wherein the at least onevalve body is depressed in the cartridge body relative to the points ofattachment and wherein the deformable material covering the at least onevalve body retains sufficient elasticity after deformation such that ina ground state the valve is open. In one embodiment the malleablematerial has undergone a plastic deformation as a result of initialvalve closure. In another embodiment the at least one valve body is aplurality of valve bodies. In another embodiment the points ofattachment comprise ridges elevated above the surface. In anotherembodiment the valve inlet and the valve outlet are fluidicallyconnected with fluidic channels. In another embodiment the malleablematerial comprises a plastic, a wax or a soft metal. In anotherembodiment the malleable material comprises a thermoplastic, athermoset, a single component resin or a multi-component resin. Inanother embodiment the malleable material comprises polypropylene,polystyrene, polyethylene, polyethylene terephthalate, polyester,polyamide, vinyl, poly(vinylchloride) (PVC), polycarbonate,polyurethane, polyvinyldiene chloride, cyclic olefin polymer (COP),cyclic olefin copolymer (COC), or any combination thereof. In anotherembodiment the deformable material comprises Santoprene a TPE(thermoplastic elasotomer) e.g., Santoprene, a blend of EPDM rubber andpolypropylene. In another embodiment the deformable material has adurometer value of between 10 Shore D to 80 Shore D. In anotherembodiment the deformable material comprises a heat seal material. Inanother embodiment a portion of the layer of deformable materialcovering a valve seat does not comprise an elastomeric material, e.g.,is not PDMS. In another embodiment the layer of deformable material hasa higher yield strength than the malleable material. In anotherembodiment the deformable material is attached to the body through anadhesive. In another embodiment the deformable material is welded to thebody. In another embodiment the deformable material comprises a materialselected from polypropylene, polyethylene, polystyrene, cyclic olefinpolymer (COP), cyclic olefin co-polymer (COC), mylar, polyacetate) and ametal. In another embodiment the cartridge comprises at least onefluidic circuit, wherein the at least one fluidic circuit comprises, aselements, at least one valve, at least one fluid inlet, at least onefluid outlet and at least one compartment, which elements arefluidically connected through fluidic channels. In another embodimentthe at least one compartment is selected from a reagent compartment, asample compartment, a mixing compartment, a reaction compartment and awaste compartment. In another embodiment at least one fluid inlet or afluid outlet comprises a via through the cartridge body. In anotherembodiment at least one compartment is a sample compartment configuredto accept a swab. In another embodiment at least one compartment ismixing chamber configured for bubbling of air through the mixingchamber. In another embodiment at least one compartment is a reactionchamber comprising a solid substrate, e.g., solid phase extractionmaterial, for retaining analyte from a sample. In another embodiment thesolid substrate comprises a material that binds nucleic acid. In anotherembodiment the solid substrate comprises Whatman paper, a carboxylatedparticle, a sponge-like material, a polymer membrane, magneticallyattractable particles, or glass particles. In another embodiment thesolid substrate binds a predetermined amount of material. In anotherembodiment the at least one fluidic circuit comprises a pump configuredas a depression in the surface. In another embodiment at least onecompartment is a reaction chamber comprising one or more thermallyconductive walls and configured for thermal cycling. In anotherembodiment at least one compartment is a waste compartment. In anotherembodiment the waste compartment comprises a material that degradesnucleic acid. In another embodiment the material that degrades nucleicacid comprises a hypochlorite salt. In another embodiment the bodyfurther comprises at least one reagent compartment comprising a reagent,wherein the compartment comprises an openable seal that, when opened,puts the compartment in fluidic communication through a via with afluidic channel on the surface. In another embodiment the deformablelayer retains sufficient elasticity to resist valve closure whennegative pressure (e.g., suction) up to about 10 psi is exerted on afluidic channel communicating with the valve. In another embodiment thevalve body and a ram configured to close the valve have shapes such thatclosure of the valve produces a pressure that is normal to the surfaceof the valve body across all areas of the valve cross section, e.g.,when the pressure of the ram is unidirectional. In another embodimentthe valve body is wider than the channel to which it is connected. Inanother embodiment the cartridge body further comprises one or morereagent compartments comprising reagents including nucleic acid primers,nucleotides and DNA polymerases sufficient to perform PCR. In anotherembodiment the reagents are sufficient for performing multiplex PCR onSTR loci. In another embodiment the cartridge further comprises achamber comprising a filter, e.g., a size exclusion filter. In anotherembodiment one of the chambers is configured as a lysis chamberconfigured to accept a biological sample, one of the chambers isconfigured as a mixing chamber configured to bubble air when liquid iscontained in the mixing chamber, and one of the chambers is configuredas a reaction chamber comprising one or more thermally conductive wallsand configured for thermal cycling. In another embodiment the cartridgefurther comprises at least one reagent compartment comprising reagentsfor performing PCR (e.g., PCR primers, nucleotides and a DNApolymerase), wherein the at least one reagent chamber comprises anopenable seal that, when opened, puts the reagent chamber in fluidiccommunication with the reaction chamber. In another embodiment at leastone reagent chamber comprises PCR primers selected to amplify aplurality of STR loci. In another embodiment one of the chambers isconfigured as a lysis chamber configured to accept a biological sample,one of the chambers is configured as an isolation chamber configured toreceive magnetically responsive capture particles and to immobilize saidparticles when a magnetic force is applied to the isolation chamber, andone of the chambers is configured as a reaction chamber comprising oneor more thermally conductive walls and configured for thermal cycling.In another embodiment the cartridge further comprises at least two setsof reagent compartments, wherein a first set of reagent compartmentscomprises reagents for performing PCR, and wherein a second set ofreagent compartments comprises reagents for performing cycle sequencing,wherein each reagent compartment comprises openable seal that, whenopened, puts the reagent compartment in fluidic communication with thereaction chamber. In another embodiment the cartridge further comprisesa reagent compartment comprising reagents to degrade PCR primers andnucleotide triphosphates. In another embodiment the cartridge furthercomprises at least two sets of reagent compartments, wherein both thefirst set of reagent compartments and the second set of reagentcompartments comprise reagents for performing PCR, and wherein eachreagent compartment comprises openable seal that, when opened, puts thereagent compartment in fluidic communication with the reaction chamber.In another embodiment the PCR reagents in one of the sets of reagentcompartments comprises reagents for performing adapted for performingquantification of human DNA. In another embodiment the cartridgecomprises a branched fluidic circuit comprising chambers connected byfluidic channels and comprising a common portion and a plurality ofbranches, wherein the common portion comprises a fluid inlet and a lysischamber, and wherein each branch comprises at least one reaction chambercomprising one or more thermally conductive walls and configured forthermal cycling, at least one isolation chamber and a fluid outlet,wherein at least the fluidic channels connecting a reaction chamber withan isolation chamber comprises a valve body. In another embodiment thecommon portion comprises a common isolation chamber. In anotherembodiment each branch further comprises at least one reagent chamberreagent compartment comprising an openable seal that, when opened, putsthe reagent compartment in fluidic communication with the reactionchamber in the branch. In another embodiment the cartridge comprises twobranches, wherein a first branch comprises reagents to perform a forwardcycle sequencing reaction on a target polynucleotide and a second branchcomprises reagents to perform a reverse cycle sequencing reaction on atarget polynucleotide.

In another aspect provided herein is an article comprising: (a) acartridge body comprising a malleable material and having, disposed on asurface of the body, at least one valve body comprising a valve inletand a valve outlet, each fluidically connected to a fluidic channel,wherein the at least one valve body comprises sloped or curved walls. Inone embodiment In another embodiment the walls are configured to exert acentering action on a tapered ram head pressed against the valve body.In another embodiment the at least one valve body is not radiallysymmetric (e.g., is longer in one dimension of the XY plane of thesurface than the other. In another embodiment the at least one valvebody does not comprise valve reliefs flanking the valve body. In anotherembodiment the at least one body does not have a flat floor. In anotherembodiment lines tangent to surfaces of opposing walls at the same depthform an acute angle. In another embodiment the at least one valve bodyhas opposing sloped walls. In another embodiment the at least one valvebody has curved walls. In another embodiment the article is configuredto exert a centering action on a tapered ram head pressed against theheat seal layer which presses against the valve body. In anotherembodiment the one or more valve bodies do not comprise valve reliefsflanking the valve body. In another embodiment the valve body has anaspect ratio of depth-to-width greater than 1. In another embodiment theone or more valve bodies do not comprise a sharp angle. In anotherembodiment the one or more valve bodies do not have a flat floor. Inanother embodiment the one or more valve bodies have a diameter betweenabout 0.2 mm (200 microns) and about 2 mm. In another embodiment theaspect ratio (width:depth) of the valve body is between about 1:1.5 and1:0.1, e.g., 1:1.2 to 1:0.7. In another embodiment lines drawn tangentto the opposing walls of the valve body at any point between the top andbottom of the valve body form an angle of at least any of is at leastany of 30°, 45° or 60° and at most any of 150°, 125° or 100°. In anotherembodiment the valve body has a depth, from its bottom to the height ofthe top of a ridge (if present) or a plane generally defined by thecartridge body surface of between 50 microns and 200 microns, e.g.,about 100 microns. In another embodiment a cross-section of the at leastone valve body has walls taking the shape of a portion of any of awedge, a circle, an ellipse, an oval, a catenary. In another embodimentthe article further comprises a relief adapted to accept a centering armfor guiding a ram head.

In another aspect provided herein is a method of making an articlecomprising: (a) providing a cartridge body comprising a malleablematerial and having, disposed on a surface of the body, at least onevalve body comprising a valve inlet and a valve outlet, each fluidicallyconnected to a fluidic channel; and (b) providing a layer comprising adeformable material; (c) bonding the layer to the surface to seal the atleast one valve body at points of attachment, thereby forming at leastone valve, wherein the at least one valve body is depressed in thecartridge body relative to the points of attachment; and (d) deformingthe deformable layer covering the at least one valve body wherein thedeformable layer undergoes plastic deformation and retains sufficientelasticity such that in a ground state the valve is open. In oneembodiment bonding comprises heat sealing or welding. In anotherembodiment deforming comprises putting mechanical pressure on thedeformable layer. In another embodiment mechanical pressure is appliedusing a ram. In another embodiment the ram has a tip having a shape thatconforms substantially to the shape of the valve body offset for thethickness of the deformable layer. In another embodiment valve body hasa cross-sectional shape that exerts a centering action on the ram towarda center of the valve body as the ram applies pressure on the deformablelayer. In another embodiment the ram comprises a flexible material. Inanother embodiment the ram is mounted on a pivot or a rocker arm. Inanother embodiment a ram assembly comprises a centering arm configuredfor coarse centering of the ram head. In another embodiment the ramcomprises a rotatable wheel mounted at an end of the ram, and whereinthe valve body has a cross-sectional shape that conforms substantiallyto the wheel offset by the thickness of the deformable layer. In anotherembodiment the ram is configured to translate laterally with respect tothe surface of the cartridge body.

In another aspect provided herein is an instrument comprising acartridge interface and a cartridge engaged with the cartridgeinterface, wherein: (I) the cartridge is a cartridge as provided herein;(II) the cartridge interface comprises: (A) at least one mechanicalactuator, each mechanical actuator positioned to actuate a valve; (B) atleast one motor operatively coupled to actuate a mechanical actuatortoward or away from a valve. In one embodiment (I) the cartridgecomprises at least one fluidic circuit, wherein the at least one fluidiccircuit comprises, as elements, at least one valve, at least one fluidinlet, at least one fluid outlet and at least one compartment, whichelements are fluidically connected through fluidic channels; and (II)the cartridge interface comprises a first port engaged with a fluidinlet and a second port engaged with a fluid outlet, and furthercomprises a pressure source configured to apply positive or negativepressure through either port to the fluidic circuit. In anotherembodiment the pressure source provides pneumatic pressure. In anotherembodiment (I) the cartridge comprises a plurality of valves; and (II)the cartridge interface comprises a plurality of mechanical actuators,wherein each mechanical actuator can be independently actuated. Inanother embodiment the instrument further comprises a source of at leastone reagent, and a pump configured to pump the reagent into a fluidinlet.

In another aspect provided herein is a method of controlling fluid flowin a fluid channel of fluidic cartridge comprising: (A) providing aninstrument as provided herein, wherein at least one of the fluidicchannels comprises a liquid; (B) closing a valve by actuating amechanical actuator against the valve to force the deformable layeragainst walls of the valve body; (C) releasing the valve by retractingthe mechanical actuator away from the valve body; and (D) moving theliquid through the valve by applying positive or negative pressure toliquid in a fluidic channel.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the disclosure are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present disclosure will be obtained by reference tothe following detailed description that sets forth illustrativeembodiments, in which the principles of the disclosure are utilized, andthe accompanying drawings of which:

FIG. 1 shows an exemplary cartridge of the disclosure.

FIG. 2 shows an exemplary cartridge body this disclosure.

FIG. 3 shows a face of an exemplary cartridge body of the disclosurewith elements of a fluidic circuit.

FIG. 4 shows an exemplary valve body of this disclosure.

FIGS. 5A-5D shows an exemplary formation of a valve of this disclosure.

FIG. 6 shows an exemplary ram head this disclosure in cross-sectionoriented toward a valve body, also shown in cross-section.

FIG. 7 shows an oblique view of a ram head positioned against a valve ofthis disclosure. The ram is mounted on a rocker arm to provide playallowing a centering action when the ram is depressed against the valve.

FIG. 8 shows a cutaway view of reagent containers of a cartridge sealedwith ball valves.

FIG. 9 shows an exemplary valve body of this disclosure.

FIG. 10 shows an exemplary ram head this disclosure in cross-sectionoriented toward a valve body, also shown in cross-section.

FIG. 11 shows an oblique view of a ram head positioned against a valveof this disclosure.

FIG. 12 shows a ram on which a downward force 3000 is applied to close avalve. The ram also has lateral play 3010 to allow centering of the ramin the valve body, promoted by turning of wheel 901 into the valve body.

FIG. 13A and FIG. 13B show two exemplary valve bodies. Tangent lines tothe walls of the valve bodies are drawn at points half way between thetop and the bottom of the valve bodies. The angle formed from thetangent lines in the valve body of FIG. 13A is about 60 degrees, whilethe angle formed from the tangent lines in the valve body of FIG. 13B isabout 130 degrees.

FIG. 14 shows an exemplary valve/ram combination.

FIG. 15 shows a cam array.

FIG. 16 shows a cam assembly comprising a ram on an axle.

FIG. 17 shows a sectioned view shows a ram pressing against a valve.

FIG. 18 shows a schematic of an example electrophoresiscartridge-comprising system.

FIG. 19 shows an exemplary system of this disclosure.

FIG. 20 shows an exemplary cartridge interface of this disclosure.

FIG. 21 shows an exemplary cartridge configuration of this disclosure.

FIG. 22 shows an exemplary cartridge configuration having a circuit withthree branches.

FIG. 23 shows an exemplary cartridge configured to perform real time PCRfor quantifying the amount of DNA in a sample.

FIG. 24 schematically illustrates an example system engaging anelectrophoresis cartridge.

FIG. 25A and FIG. 25B are isometric view of an example electrophoresiscartridge.

FIG. 26 shows a schematic of an example electrophoresis cartridge.

FIG. 27A and FIG. 27B are isometric views of the system of FIG. 19.

FIG. 28 shows a sequence of steps in sample preparation. “O” indicates aram does not press against a valve. “X” indicates a ram presses againstand closes a valve.

FIG. 29 shows an alternate exemplary ram of this disclosure.

DETAILED DESCRIPTION OF THE INVENTION I. Introduction

This disclosure provides, among other things, a valve for a fluidicdevice, such as a mesofluidic or microfluidic device. The device can bea cartridge adapted to engage a cartridge interface that operatesactivities within the cartridge. Alternatively, the device can be afluidic device comprising the valve and included as an integral part ofa system that performs fluidic operations. The fluidic device cancomprise a substrate having a surface with fluidic elements disposedthereon, and a layer of deformable material covering the fluidicelements. For example, the device can be in the form of a chip or acartridge.

The cartridge can comprise a cartridge body having a valve body disposedtherein, and a flexible or semi-flexible layer bonded to a surface ofthe cartridge body. The valve is comprised within the combination of thecartridge body and the layer. The valve includes a valve inlet and avalve outlet, typically communicating with fluidic channels. The valvecan be closed by pressing the layer against a valve seat of the valvebody, thereby obstructing the flow of fluid through the valve. Pressurecan be applied mechanically, for example with a ram. The valve can beopened by releasing the pressure against the layer. In certainembodiments, the valve is self-opening. In other embodiments, the valveis initially opened by application of force, for example, forcing liquidthrough a closed valve. In either case, once open, the layer hassufficient elasticity to pull away from the valve seat withoutapplication of external force, such that the valve is biased in an openstate. This is also called a “normally open” valve, or a valve open inthe “ground state”. Such a valve resists closure when negative pressures(e.g., vacuums) are applied through a valve inlet or outlet at pressurestypically used to move liquids in mesofluidic or microfluidic devices.

As used herein, the term “nanofluidic” refers to a passage having anaspect no greater than 500 microns. As used herein, the term“microfluidic” refers to a passage having an aspect no greater than 1000microns. As used herein, the term “mesofluidic” refers to a passagehaving an aspect no greater than 1500 microns. As used herein, the term“macrofluidic” refers to a passage having an aspect greater than 1500microns. Ranges between these limits are also contemplated, e.g.,between 500 microns and 1500 microns or, greater than 500 microns.

II. Fluidic Device

A. Configuration

Referring to FIG. 1, exemplary cartridge 100 comprises a cartridge body101 backed by a deformable layer 102. In this embodiment, body 101 ismade of injection molded polypropylene. Deformable layer 102 is a layerof heat seal material which is a laminate including a polypropylenelayer on a polyethylene backing. Stoppered compartments filled withreagents open on ports 114, 115 and 116.

FIG. 2 shows a side of body 101, including ports 111, 112, 114, 115 and116; and walls of chambers 120, 121 and 123. Positive or negativepressure can be applied to the fluidic circuit through port 112 to moveliquids in the fluidic circuit.

FIG. 8 shows an exemplary cartridge having ports 114, 115 and 116configured as chambers communicating with fluidic channels through vias.The chambers are configured to contain reagents and to deliver reagentsto a fluidic circuit. The reagent delivery member is shown here as adual plunger sealed chamber 116 having a first stopper 801 and a secondstopper 802, in this case configured as balls. The stoppers 801 and 802seal a receptacle (e.g., a column or tube of port 116) having a reagent(e.g., premix, master mix), such as an STR master mix. The applicationof force to the first stopper ball 801 (such as, e.g., with the aid of aplunger or a syringe) actuates the movement of the second stopper ball802 ball away from the via, creating a flow path for the reagent to passinto channels 131 and 134.

FIG. 3 shows the fluidic side of exemplary body 101. Fluidic elements ofthe cartridge can be formed on a substrate having a surface that issubstantially flat and that defines a plane. Various elements of afluidic circuit are formed in the surface of the substrate by methodsknown to one skilled in the art, e.g., injection molding, hot embossing,laser cutting and 3D printing. Elements may be disposed below the planeof the surface, e.g., as channels, wells or chambers, or valve seats.Other elements, such as ridges, can protrude above the plane of thesurface.

Cartridge body 101 can include closable cap 164 to close sample/lysischamber 1120.

Wells formed in the cartridge body create chambers 120, 121, 122, 123and 130. Cartridges can comprise at least one fluidic circuit whichcomprises, as elements, at least one fluid inlet, at least one fluidoutlet, at least one valve, and at least one compartment, which elementsare fluidically connected through fluidic channels. Fluidic elements,such as channels, chambers and valve bodies, typically includedepressions in the body surface. A via (a passage from one side of acartridge body to another) is distinguished from a depression, which isformed in, rather than through, the body. Inlets and outlets can beformed as vias or openings at an edge of the fluidic side of thecartridge, e.g., as the termination of a channel, e.g., into acompartment such as a sample chamber.

Cartridge body 101 can include sample/lysis chamber 120. Sample/lysischamber 120 is configured to accept a swab, punch, or other sample type.This compartment can also serve as a lysis chamber. To accommodate theswab, punch, or other sample type, it can have a volume ranging from,e.g., 10 μL to 15 mL, e.g., 250 μL to 1 ml. Cells are lysed andanalytes, such as DNA, RNA or protein, can be extracted from the swab,punch, or other sample type in this chamber.

Cartridge 101 can include reagent chambers (e.g., 114, 115 and 116)filled with, e.g., nucleic acid size standards (molecules of knownsizes), PCR master mix and PCR primers, respectively, and sealed with,e.g., balls (801, 802) acting as closures for ball valves. When opened,the reagent chambers come into fluidic communication with fluidicchannels in sample cartridge 101, for example, through ports 152(internal lane standard), 153 and 154 (PCR Master Mix and PCR PrimerMix). Pistons can actuate the ball valves, pushing fluids through theports and into the channels to which they are connected. Samplecartridge 101 also can include inlet port 112 and output port 113. Uponengagement with the cartridge interface, inlet port 112 and outlet port113 each engage a fluid line. The fluid line connected to inlet port 112can be attached to a pressure source, e.g., a syringe, to exert positiveor negative pressure to fluidic channels via the inlet port,transporting liquids, such as lysis buffer, water or air, into or out ofthe cartridge. The fluid line connected to output port 113 can conductanalyte from the cartridge to a sub-system for analyte analysis.

Exemplary cartridge 101 includes pump 184. Pump 184 (e.g., an air pump)is configured as a chamber defined by walls of the cartridge body. Pump184 is fluidically connected to at least one fluidic channel in thecartridge body. Walls of the pump comprise, at least in part, themalleable material of the cartridge body. Accordingly, the walls can bedeformed, for example by mechanical force, increasing pressure in thechamber to pump liquid or air in fluidic channels in fluidiccommunication with the pump. Pump 184 can be actuated with a plunger orpiston that depresses walls of pump 184 and forces, for example, airfrom the pump body through the fluidic channel to which it is connected.Pump 184 can be used to clear fluid from a fluidic channel. For example,in this embodiment, reagent introduced from port 152 into reactionchamber 122 may leave dead volume in channel 131. Pump 184 can be usedto pump this dead volume of reagent into reaction chamber 122.

In some embodiments, the cartridge comprises a filter to filter liquidfrom a biological sample, such as a cell lysate. One such embodiment isshown in FIG. 3. The exemplary cartridge comprises filter chamber 130comprising a filter, in a flow path between lysis chamber 120 andreaction chamber 122, and typically between the sample chamber and afirst valve, e.g., 146. The filter can be a size exclusion filter havingpores no greater than any of 100 microns, 50 microns, 25 microns, 10microns or 5 microns. The filter can be, for example, a mixed celluloseester, such as a Millipore™ Membrane Filter. Such a filter can be usefulto capture particles that may clog valves or chambers in the flow path.In another embodiment, the filter is included around the outlet of thelysis chamber.

Chamber 121 functions as a mixing chamber. A mixing chamber can have atapered shape such that air delivered from a channel on a side towardgravity bubbles air toward a side away from gravity.

Chamber 122 functions as a reaction chamber. This chamber can betemperature regulated, for example, to perform PCR. The reaction chambercan serve to capture DNA or house a small amount of lysate for directamplification. It can also be used for cleanup and amplification. Tominimize the duration of thermocycling and the amount of energyrequired, this chamber should have minimal volume, perhaps ranging from2 μl to 25 μl, although other configurations are practical.

In one embodiment a fluidic device of this disclosure comprises areaction chamber that comprises a solid substrate, e.g., solid phaseextraction material 186, for retaining analyte from the sample. Thesolid substrate can comprise a material that binds the analyte, such asa nucleic acid such as DNA. The amount of solid substrate in a chambercan be selected to retain the predefined amount of analyte. For example,the material can be Whatman paper or carboxylated particles.Alternatively, the solid substrate can be an absorbent or sponge-likematerial that absorbs a predetermined volume of fluid. The material canbe in the form of a monolith. The material can be, for example, PVDF(polyvinyldiene fluoride) or other membranes, filter paper, glass fiberfilters, magnetically attractable particles, chromatography media, solidphase extraction materials, or glass particles. In operation, lysate ispumped through the chamber and a predetermined amount of analyte isretained on a solid substrate. Then, retained material is contacted withreagents, e.g., reagents for PCR. The resulting material can beincubated to form a reaction product. For example, the chamber can beput into thermal contact with a thermal-control device, such as aPeltier, and the reaction mixture can be thermally cycled. In anotherembodiment, the chamber can include a pocket or container designed toretain a defined volume of liquid. In another embodiment, the chambercan have a surface coating that retains the desired analyte, e.g., anantibody to capture epitopes or a single stranded nucleotide to capturea target nucleic acid. In another embodiment, the chamber could have acoating, e.g., PEG, that will retain a desired volume of liquid.

Chamber 123 functions as a waste chamber. A waste chamber can containmaterial 185 that degrades nucleic acids, polypeptides, or otheranalytes. For example a material can comprise a chlorinated material,such as calcium hypochlorite. In another embodiment, the material cancomprise an enzymatic activity such as a DNAase, RNAase, protease, etc.Alternatively, the waste chamber can include an absorbent material thatabsorbs waste containing liquid. In another embodiment the nucleic aciddegrading material is contained in a water-soluble capsule. In yetanother embodiment the nucleic acid degrading material is combined withan absorbent material such as cellulose or polypropylene fibers.

These functional elements are fluidically connected by fluidic channels,for example, channels 130, 131, 132, 133 and 134. Liquid flow along thechannels is regulated by valves. The valves include valve bodies formedin the cartridge body and portions of the deformable layer covering thevalve body that can be deformed into the valve body, blocking flow inthe fluidic channel. Valve bodies are shown in FIG. 3 at variouspositions, e.g., 141-149. More specifically, these valves include ventvalve 141, waste vent valve 142, product top valve 143, product bottomvalve 145, lysis valve 164, lysis transfer valve 147, cycler in valve148, cycler out valve 149, product out valve 144.

Cartridge body 101 includes the following valve bodies: 141 Cycler Out(A0), 142 Lysis (A1), 143 Waste Shut Off (A3), 144 Waste In (A4), 145Cycler In (B0), 146 Lysis Transfer (B1), 147 Product Bottom (B2), 148Product Top (B3) and 150 Vent (B4).

The valve is configured so that after initial closure and removal of theclosing force, the valve is biased open (e.g., “normally open”). Thischaracteristic is a function of the valve geometry and the properties ofthe deformable layer. In general, the deformable layer is attached tothe valve body at a position that is above the bottom or floor of thevalve. In this configuration, a deformable material that undergoeselastic, but not plastic, deformation upon valve closure will return toa position that leaves the valve open after the deforming force isrelieved. In situations typical of fluidic devices in microscale ormesoscale range, this may be the case for elastomeric materials such asPDMS. However, materials such as heat seal, used as a deformablematerial in some embodiments of a cartridge, are expected to undergoboth elastic and plastic deformation when deformed into a closed valveposition. In this case, the valve geometry can be selected such thatafter deformation on valve closure, the material retains sufficientelasticity so that after release of pressure closing the valve, thedeformable material returns to a shape that leaves the valve biasedopen.

FIG. 4 is a detailed view of a valve body, e.g., element 141. Fluidicchannel 133 includes raised ridges 151 and 152. These ridges are raisedabove the plane (i.e., away from the body) of the generally flat surface103 of the fluidic side of the cartridge body 101. Furrow 154 of channel133 forms a recessed channel in surface 103 of the fluidics side, thatis, disposed below the plane (i.e., into the body) of surface 103. Incertain embodiments, valve bodies do not have valve reliefs flanking thechannel walls. A valve with deformable material attached is shown, e.g.,in FIG. 5.

In another embodiment surface 103 can comprise a well positioned nearthe valve body. Such a well can be used as a guide for a pin to providecoarse alignment of the ram head before depressing the ram head into thevalve body.

Materials for the cartridge body and the deformable layer are selectedwhich can be deformed by pressure exerted by the source of positivepressure, e.g., a mechanical ram. Typically, the deformable layer hasthe ability to stretch under pressure, and the valve body has theability to compress or flow under pressure. In this way, concentratedpressure can be exerted against the valve sufficient to compressportions of the valve body without breaking (e.g., cracking or piercing)the deformable layer. Combinations of materials for the cartridge bodyand the deformable layer include, for example, polypropylene and heatseal, polyethylene and laser-welded polyethylene and Teflon and aluminumfoil bonded with a patterned adhesive.

In order to initially close the deformable layer against the valve seat,the ram applies an appropriate pressure against the deformable layer inthe appropriate position. In certain embodiments, valve bodies have across section that progressively narrows toward the bottom of thedepression forming valve seat of the valve body.

Several factors can be considered in configuring the valve. In certainembodiments, as regards the aspect ratio of the cross-section(width:depth), the valve body has sufficient depth such that the bottomof the valve body is disposed deeper in the valve body than the pointsof attachment of the malleable layer. In fact, the deformable layer maybe attached at a point on a ridge disposed above the plane of thesurface of the cartridge body. In this configuration, a deformable layerof heat seal will experience elastic and plastic deformation wheninitially depressed, but retain sufficient elasticity to return, atleast partially, to its original shape, leaving an aperture in thevalve. Accordingly, the valve, in its ground state, is normally open, orbiased toward open. In this configuration, the aperture is sufficientlylarge to allow passage of particulate material in a liquid sample passedthrough it.

In other embodiments, the valve is configured so that the deformablelayer retains sufficient elasticity to resist valve closure whennegative pressure (e.g., suction) is exerted on a fluidic channelcommunicating with the valve. For example, the valve can resist closureupon application of negative pressures up to about 10 psi.

The aspect ratio (width:depth) of the valve body can be between about1:1.5 and about 1:0.1, e.g., about 1:1.2 to about 1:0.7.

Valve bodies of the devices of this disclosure typically have a diameterbetween about 0.2 mm (200 microns) and about 2 mm.

In certain embodiments, valve body is not so narrow (which is to say,does not have an aspect ratio (width:depth) that is so small) that thedeformable layer breaks (e.g., tears or is punctured) when pressedagainst the valve body by a mechanical ram. The appropriate depth can bedetermined by the ordinarily skilled artisan based, in part, on thepliability of the deformable layer.

In another embodiment, the valve body has walls having a configurationto provide a centering action against a ram pressed against deformablelayer on the valve body. The ram may be off-center with respect to thecenter line of the valve body in cross section. This configuration canbe useful if the size of the valve is sufficiently small that there islittle tolerance for the ram head to be off-center with respect to thevalve body.

In one aspect, lines drawn tangent to level points of opposing walls ofthe valve body at any point between the top and bottom of the valve bodyform an angle between any of at least 30°, at least 45° or at least 60°and any of at most 150°, at most 125° or at most 100°.

The valve body can have depth, from its bottom to the height of the topof a ridge (if present) or a plane generally defined by the cartridgebody surface of between 50 microns and 200 microns, e.g., about 100microns.

Many valve geometries are consistent with the use of heat seal as adeformable material. In one embodiment, the diameter of the valve bodydecreases with increasing depth. However, the aspect ratio of the valveshould not be so narrow that closure of the valve results in failure ofthe deformable material, such as piercing or tearing. In anotherembodiment, the valve body and the ram have shapes such that closure ofthe valve produces a pressure that is normal to the surface of the valvebody across all areas of the valve cross section. In some embodiments,this normal pressure is exerted when the pressure of the ram isunidirectional, e.g., only in a vertical direction or normal to theplane defined by a surface of the cartridge body into which the valve isdisposed.

Within these parameters, the cross-section of the valve body can take avariety of shapes. In one embodiment, the cross section is substantiallywedge shaped (e.g., a triangle with a curved radius at the bottom, e.g.,with a blunt rather than sharp point at the end). In another embodimentcross-section takes the shape substantially of a catenary or a parabola.In another embodiment, the cross-section is substantially circular. Ifthe cross section of the valve has the shape of a cord of a circle, theincluded angle of the chord can be between about 30° and 60°.

In an exemplary fluidic device, the body can comprise a polymer, e.g.,polymer is a polycarbonate, an olefin co-polymer (COC) (e.g., Zeonor), acycloolefin co-polymer (COP), an acrylic, a liquid crystal polymer,polymethylmethoxyacrylate (PMMA), a polystyrene, a polypropylene, or apolythiol. The deformable layer can comprise a siloxane, such as PDMS.The deformable layer can be bonded to the body by coating the bodysurface with hydroxyl groups and binding the deformable layer throughthe oxide groups. In one embodiment, the polymer body is coated with anoxide, e.g., a metal oxide (e.g., aluminum oxide or titanium oxide) or asemiconductor oxide (e.g., silicon oxide or germanium oxide). Suchbonding is described in, for example, U.S. Pat. No. 8,584,703.

B. Materials

-   -   1. Cartridge Body

The body can have an external surface comprising elements of fluidiccircuits, such as channels, compartments, vias, and valve seats. Thebody can be made by injection molding of the thermoplastic, 3D printingor other methods well known to one skilled in the art. These featurescan be covered with a layer of deformable material attached to thesurface of the cartridge body. The layer can function to seal otherwiseopen features such as channels and compartments. The layer of deformablematerial can deform to contact a valve seat, thereby closing the valve.

The material can be attached to the surface of the body using aselective bonding process in which the material bonds to selectedportions of the surface during the bonding process and does not bond toun-selected portions of the circuit after the bonding process iscomplete. For example, the material may bond to surfaces other thanfluidic elements during the bonding process, and not bond to fluidicelements, such as channel floors, chamber walls and valve seats, afterthe bonding process. Methods for selective bonding include, for example,thermal bonding (e.g., heat sealing, welding, laser welding), chemicalbonding (e.g., chemical bonding of oxide to PDMS, vapor bonding) andselectively placed adhesives (adhesive bonding). In certain embodiments,bonding is not thermal bonding and/or is not chemical bonding and/or isnot adhesive bonding.

In one embodiment a layer of the deformable material is attached to asurface of a cartridge body through thermal bonding. This can includethermally bonding the material directly to the surface, or thermallybonding the material through an intermediate layer of material. In thelatter case the material can be a laminate in which a deformablematerial is coated with a layer of material that contacts the surfaceand that melts at lower temperature than the deformable material. Ineither case bonding typically comprises contacting the layer ofdeformable material to the body to form a combination and using a die toapply heat and pressure to the combination. Application of heat andpressure melts substrates in locations at which the material and bodyare in contact and fuse them, e.g., through coalescence. This process ismore generally referred to as welding.

At least the valve body of the cartridge body can comprise a malleablematerial, that is, a material capable of plastic deformation. Thematerial, after being deformed, does not return to its original shape.In certain embodiments the malleable material is a plastic, a wax or asoft metal (e.g., lead). The plastic can be, for example, athermoplastic, a thermoset, a single component resin or amulti-component resin. In one embodiment, the cartridge can comprise aninjection molded body, for example, a thermoplastic, and a deformablelayer bonded to the body. The thermoplastic can include anythermoplastic known to those skilled in the art, such as polypropylene,polystyrene, polyethylene, polyethylene terephthalate, polyester,polyamide, vinyl, poly(vinylchloride) (PVC), polycarbonate,polyurethane, polyvinyldiene chloride, cyclic olefin copolymer (COC), orany combination thereof.

-   -   2. Layer of Deformable Material

The layer of deformable material (also called a “deformable layer”) usedin cartridges disclosed herein can comprise a plastic material (plasticdeformation) or an elastic material (elastic deformation). The plasticmaterial can comprise, without limitation, a polymer or a metal.Suitable plastic materials for the layer of deformable material include,without limitation, polypropylene and polyethylene. Suitable metalsinclude, for example, aluminum. Suitable elastic materials include, forexample, elastomeric materials such a polysiloxanes, e.g., PDMS. Otherdeformable materials are further described herein. In certainembodiments, the deformable material is not an elastomeric material. Asused herein an “elastomer” or “elastomeric material” is a material, suchas a natural rubber or a synthetic rubber (e.g., a silicone (such asPDMS) or Santoprene), having a low Young's modulus. Such materials, usedin the microvalve configurations described herein, undergo elastic, butnot plastic, deformation.

A material that bonds to a body through application of heat and pressureis referred to herein as a “heat seal”. Heat seals are well known in theart and are commercially available. For example, 4titude (Wolton,Surrey, UK) commercializes a variety of heat seals. These heat seals aredescribed on the www website 4ti.co.uk/sealing/heat-seals/. Theseinclude, for example, Clear Seal, Clear Weld Seal, and Foil Seal. Heatseals also are produced by Axygen, a Corning brand (Corning, Tewksbury,Mass., USA). These include Axygen® PlateMax heat sealing film andsealing film rolls. See the website:catalog2.corning.com/LifeSciences/en-US/Shopping/Product.aspx?categoryname=Genomics+and+Proteomics(Lifesciences)%7cPCR+Products(Lifesciences)%7cSealing+Films+and+Tapes+for+Microplates(Lifesciences)%7cHeat+Sealing+Films+and+Tapes+for+Microplates(Lifesciences). Such materialsexhibit partially elastic properties, that is, their deformation ispartially reversible.

The deformable material can be a homogenous or non-homogenous material.In certain embodiments, the heat seal material is made from the samematerial as the body of the cartridge. It can comprise a thermoplastic(e.g., polypropylene, polyethylene, polystyrene, cycloolefin co-polymer(COC), mylar, polyacetate) or a metal (e.g., aluminum). See, e.g., WO2012/136333. The heat seal can be produced by contacting a heat seallayer with the body and applying heat and pressure. Non-homogenous filmsinclude laminates having a first side for contact with the heater and asecond side for contact with the body. The first side has higher meltingtemperature (“high melt”) than the second side (‘low melt“). Thispermits use of a heat source to bring the second side to its meltingtemperature before the first side allowing bonding to the body withoutbonding to the heater.

III. Method of Making

FIGS. 5A-5D show the attachment of a layer of deformable material to thefluidics face 103 of cartridge body 101 and the formation of a closedvalve. FIG. 5 depicts a cross-section of valve 141.

FIG. 5A shows cartridge body 101, heatable die 105 and laminated seal102 sandwiched between them. Cartridge body 101 includes surface 103,ridges 151 and 152 (optionally), and groove 171. This figure also showsthat ridges 151 and 152 are formed as areas raised against the planarsurface 103, while groove 171 is formed as an area depressed or recessedagainst the plane of surface 103.

Ridges can be between about 25 microns and about 100 microns above theplane of the surface of the cartridge that mates with the deformablelayer, e.g., about 50 microns. The groove of the valve body can have adepth below the plane between about 50 microns and about 300 microns. Ifthe deformable layer is attached by a method other than heat sealing,e.g., with a patterned adhesive or with laser welding, then the ridgesdo not need to rise above the plane of the surface. Fluidic channelsthat are either microfluidic, that is, having an aspect less than 500microns) or macrofluidic, that is, having no aspect less than 500microns (e.g., having no aspect less than 1000 microns). Fluidicchannels can have a depth of about 100 microns to about 400 microns.

The layer of deformable material 102 is bonded to cartridge body 101 bypressure and heat applied by die 105. The layer of deformable material102 is, in this example, a laminate. One side of the laminate contactsthe die. This side has a melting temperature higher than the temperatureof the die. Therefore, when the heated die is pressed against thedeformable material 102 the higher melting side does not melt or stickto the die surface. The other side of the laminate contacts surface 103of cartridge body 101 and has a melting temperature lower than thetemperature of the die. Accordingly, this material melts when the heateddie is applied. The cartridge body also comprises a malleable materialthat melts when pressed against the heated die.

Consequently, as shown in FIG. 5B, after application of pressure andheat by the die, ridges 151 and 152 are compressed or crushed. Also, thelayer of deformable material is selectively bonded to the cartridgebody, in this case, to surface 103 and to areas of attachment on thevalve body, e.g., to ridges 151 and 152. Through this bonding thedeformable layer seals fluidic elements of the fluidic circuit,including ports, chambers, channels and valves.

FIG. 5C shows closing of the valve in cross-section. Ram head 602 has ashape that conforms substantially with the shape of groove 171.

The oblong shape of the valve body allows for the ram head to be shiftedalong the axis depicted by the circle X. Accordingly, in an instrumentin which a plurality of rams in a cartridge interface must be alignedwith a plurality of valves in a cartridge engaged with the interface,flexible rams and the self-centering action of the groove providegreater tolerance for the relative positioning of rams and valves.

The pressure of the ram presses the layer of deformable material againstthe valve body and, in an initial application, stretches the deformablematerial causing plastic deformation of the deformable layer. This coinsor stamps the valve body into a closed or closable position. However,the valve body is configured with a shape such that, even afterstretching, the deformable material retains some elasticity. For thispurpose, the layer of deformable material must have a strength (e.g.,yield strength or ultimate strength) that is greater than the strengthof the ridges, such that pressure from the ram crushes the ridges butdoes not break the deformable layer. In this configuration the valve isclosed and no liquid can pass through the valve.

Referring to FIG. 5D, to open the valve to allow the passage of liquid,the ram is retracted from the cartridge. The deformable layer is bondedto the remains of the ridges, to the edges of the valve body or to upperparts of the wall of the valve body. But the deformable material is notbonded to the floor of groove 171. Due to the slope of the walls of thevalve body, the deformable layer retains sufficient elasticity to“spring back” from the floor of the groove. This produces a passage 175through the valve through which liquid can pass. Thus, there is formed avalve that is biased open after pressure in the channel is released. Toclose the valve, ram head 602 is pressed against the layer of deformablematerial, actuating the layer of deformable material against the valveseat and closing the passage, as shown in FIG. 5C. Accordingly the valveseat comprises those portions of the valve body with which thedeformable material comes into and out contact.

Form and function of the valve improve when the ram head is centeredwith respect to the valve body. Such an orientation promotes a tightseal between the malleable material and the valve body. In certainembodiments the ram and valve exhibit a self-centering action. The ramcan be mounted on a pivot or have a flexible or swingable shaft thatallows for lateral displacement of the ram as is it pressed into thevalve body. When the walls of the valve body are sufficiently steep, aram pressed against the deformable material overcomes the force offriction to slide toward the valve body midline. Alternatively, the ramcan include a centering arm configured to fit in a centering well ordepression in the cartridge body. Such a centering arm can providecoarse centering for the ram head against the valve body.

Valves are closed by use of force applied by a ram. FIG. 6 shows anembodiment of the head of ram 601 having a wedge shape that conformssubstantially to the shape of valve body 141. Ridges 151 and 152protrude above the plane of surface 103. Heat seal material 102 overlaysthe cartridge body. A fluidic channel into which the valve leads isdepicted by lines 621 and 622. FIG. 7 shows ram head 601 orientedagainst valve body 141 in oblique view. Valve body 141 includes raisedridges 151 and 152. Channel 620 also includes raised ridges 621 and 621which can promote bonding of the deformable layer and sealing of thechannel and the valve.

FIG. 14 shows an exemplary valve/ram combination. Centering guide 1402has a tapered head and mates with well 1401 to provide coarse centeringfor ram head 602 into a valve body defined by ridges 151 and 152 andfurrow 171. This configuration can be used with the ram actuationassembly depicted in FIG. 15, FIG. 16 and FIG. 17. FIG. 29 shows analternative exemplary embodiment of a ram. Ram head 2901 closes a valveof this disclosure. Ram head 2901 includes wings disposed lateral to thevalve and in line with the flow path. These protrusions function toreduce stress on the valve and tearing of the deformable material.Centering guide 2902 has a tapered head for mating with a centeringwell.

In another embodiment, friction of the deformable material againstcentering by the ram is overcome by providing a ram having a head in theform of a rotatable wheel. The valve body can have a cross-sectionalshape that conforms to the shape of the wheel, such as substantiallycircular. Such an embodiment is shown in FIG. 9, FIG. 10 and FIG. 11.Valve body 941 Includes ridges 951 and 952 which protrude above theplane of surface 903. A fluidic channel 920 into which the valve leadsalso includes raised ridges 921 and 922. Heat seal material 102 overlaysthe cartridge body. The shape of the ram head conforms substantially tothe shape of the valve body. FIG. 10 shows a cross-section of awheel-shaped ram disposed over a valve body. Ram head 901 is configuredas a wheel mounted on spindle 930 and rotatable by use ball bearing 940.Ram 950 on which the ram head is mounted comprises a centering spring toallow the ram to move laterally and to be centered in the valve body.FIG. 11 shows ram head 901 oriented against valve body 941 in obliqueview. As the ram is pressed into the valve body, the wheel rolls towardthe centerline, producing a tight seal.

IV. Cartridge Interface and Operation

FIG. 15, FIG. 16 and FIG. 17 show an embodiment of a cartridge interface1500 mated with a sample cartridge 1801. In this embodiment rams arecomprised in a cam mechanism 1300. Cam mechanism 1300 comprises cam 1313and cam follower 1311. Cam 1313 has cam surface 1321, cam protrusion1319 and cam aperture 1315 configured with a cam key 1317 adapted toposition the cam on a rotating element. Cam follower 1311 is mounted onlever 1303, which rotates about an axle connected through aperture 1301.Ram 1309 (sometimes referred to as a “knife”) is configured as aprotrusion mounted on lever 1302. Ram 1309 can be configured as a pieceheld in place by a holder on lever 1302. Ram 1309 can comprise centeringguide 1333 and ram head 1331, adapted to press into the valve. The levercan be biased toward the valve with a biasing element 1305, for examplewith a spring. A spring can be mounted on mount 1304. In one embodiment,a biasing element exerts a force of at least any of 5 pounds, 6 pounds,7 pounds, 8 pounds, 9 pounds or 10 pounds. The cam can be a plate cam,e.g., can take the shape of an eccentric disc comprising an eccentricity1319. The cam can include an aperture 1317 adapted for mounting the camon a rotation device, such as a drum driven by a motor. The cam aperturecan include a key 1317 to align the cam on the drum. Rise of the camfollower pushes the ram away from the valve. Return of the cam followerallows the ram to push against the valve from the force of the biasingelement. In certain embodiments, the cam surface 1321 may pull away fromcam follower during valve closure, to allow biasing element 1305 toexert full force against the valve.

Cartridge interface 1500 includes ram actuation assembly 1517, baseplate 1515 and cover plate 1516. Sample cartridge 1801 is inserted intoa slot formed by the mating of base plate 1515 and cover plate 1516.

Ram actuation assembly 1517 can include cam array 1501; ram array 1509and an array of biasing elements. The cam array can comprise a pluralityof cams 1313 mounted on rotating drum 1507. The ram array can comprise aplurality of ram effectors, each comprising lever 1303 mounted on axlethrough aperture 1301, ram 1309 and cam follower 1311. Each rammechanism is configured to engage a cam, and each ram to engage a valveof the sample cartridge.

Referring to FIG. 20, base plate 1515 includes a surface against which asurface of a cover plate can mate and apertures through which rams canprotrude. Rams can be biased against a wall of the aperture forguidance. A cover piece 2016 mates with a face of base plate 1515 andforms a guide. The guide guides a rail on cartridge 1801 when thecartridge is inserted into the cartridge interface. A top piece 2017includes apertures for pistons or plungers configured to actuate ballvalves to chambers connected to ports. These include pistons 2042, 2045,2046 and 2050, which are aligned with valves and which provide a stopagainst the pressure of the rams. Apertures 2014, 2015 and 2016 guideplungers to depress ball valves. The top piece also includes apertures2012 and 2013 through which fluid conduits pass to mate with input andoutput ports in the cartridge. The conduits are fluidically connected toa sample analysis sub-assembly, to convey analytes and air pressure tothe cartridge.

Turning the drum through a rotation closes and releases valves when theeccentricity engages and disengages the cam follower. In one embodiment,the eccentricities on the drum are staggered, such that during arotation of the drum valves are closed in a pre-determined sequence. Theeccentricities can be staggered, for example, by positioning theeccentricity relative to the key such that mounting the cam on the drumplaced the eccentricity in a pre-selected position. One such sequence isshown in FIG. 28. The sequence is executed by one full turn of the drum,which can be turned by a stepper motor to each next position in therotation.

V. Integrated System

Systems provided herein may be capable of preparing, processing andanalyzing a single sample or a plurality of samples. Several operationscan be performed by the system provided herein, for example, (a)receiving one or more samples; (b) isolating and extracting targetmaterial from the received sample; (c) purifying and amplifying thewhole target material or selective portion of the target material toproduce an analyte ready to be examined; and (d) separating, detectingand analyzing the prepared analyte. These operations can be conductedand performed in a system that comprises several integrated sub-systems,for example, a sample preparation sub-system, a sample analysissub-system and a control-sub-system. In some cases, a system maycomprise a user interface, a sample cartridge interface, and anelectrophoresis interface. The sample cartridge interface and theelectrophoresis interface are configured to releasably engage with asample cartridge for sample processing, and an electrophoresis cartridgefor sample analysis respectively.

Systems provided herein can be fully automated, enabling a user toreceive, process and analyze a sample without substantial labor andinput. Sample preparation, processing and analysis can be accomplishedin provided systems without the necessity of manually removing andtransferring the sample, reagents and analytes among different parts inthe system. Since the incorporated sub-units (e.g., sample cartridge andelectrophoresis cartridge) are highly integrated and bear small sizes,systems provided herein can be dimensioned to minimize footprint,enabling the portability and usefulness in a wide context ofapplications. For example, the systems may be used in on-the-gosituations, such as remote locations, or they may be used in situationsin which transportation is not readily available or user mobility isdesired, such as battlefields scenarios and crime scenes.

The cartridges of this disclosure are useful in integrated and automatedsample-to-answer systems that, starting from a sample comprisingbiological material, generate an analysis of the sample. In otherembodiments, the cartridges can be used for stand-alone samplepreparation. In certain embodiments, the biological material is DNA andthe genetic profile involves determining one or a plurality of allelesat one or a plurality of loci (e.g., genetic loci) of a subject, forexample, a STR (short tandem repeat) profile, for example as used in theCODIS system. The system can perform several operations, including (a)extraction and isolation of nucleic acid; (b) amplification ofnucleotide sequences at selected loci (e.g., genetic loci); and (c)detection and analysis of amplification product. These operations can becarried out in a system that comprises several integrated modules,including an analyte preparation module; a detection and analysis moduleand a control module.

Various chemistries are commercially available to perform STR analysisand, in particular, CODIS-compatible STR analysis. These include, forexample, Globalfiler® and Globalfiler® Express (6-dye, 24-locus STR kit,from Life Technologies/Thermo Fisher Scientific (Grand Island, N.Y.)(worldwide web site:lifetechnologies.com/us/en/home/industrial/human-identification/globalfiler-str-kit.html), and PowerPlex® Fusion (e.g., PowerPlex®Fusion 6C) from Promega Corporation (Madison, Wis.) (worldwide web site:promega.com/Products/Genetic-Identity/STR-Analysis-for-Forensic-and-Paternity-Testing/PowerPlex-Fusion-STR-Kits?utm_medium=print&utm_source=ishi_poster&utm_campaign=powerplex&utm_content=October).

Systems provided herein may be fully integrated. Sample processing canbe accomplished in a single system without having to remove a sample andtransfer it to another system. Systems provided herein can be fullyautomated, enabling a user to process a sample without substantial inputfrom the user.

FIG. 19 shows an exemplary system of this invention. System 1900includes several functional elements. System 1900 can include a samplepreparation sub-system, a sample analysis sub-system and a controlsub-system. Such an instrument comprising an interface for engaging acartridge of this disclosure, actuating valves and moving liquids isdescribed in U.S. Provisional Patent Application Ser. No. 62/069,752,filed Oct. 28, 2014, which is incorporated herein by reference in itsentirety.

A sample preparation sub-system can include a sample cartridge interface1500 configured to engage a sample cartridge 100, sources of reagentsfor performing a biochemical protocol, a fluidics assembly configured tomove reagents within the sample preparation sub-system. A fluidicsassembly can include a pump, such as a syringe pump. The pump isfluidically connectable through valves to the outlets for reagents suchas water and lysis buffer and to a source of air. The pump is configuredto deliver lysis buffer and water through fluidic lines 1910 and 1911,respectively, to inlet port 112 in the sample cartridge. Air or liquidpressure applied by the pump to inlet port 112 can pump analyte outoutlet port 113 and through line 1912 into the analyte inlet in theelectrophoresis cartridge.

A sample analysis sub-system can include an electrophoresis assemblyincluding an anode, a cathode and an electrophoresis capillary inelectric and fluidic communication with the anode and cathode, and asample inlet communicating between a sample outlet in the samplecartridge and an inlet to the capillary. These can be contained, e.g.,within an electrophoresis cartridge 1905. The sample analysis sub-systemcan further include an optical assembly including a source of coherentlight, such as laser 1988, an optical train, including, e.g., lenses1955, and a detector, configured to be aligned with the electrophoresiscapillary and to detect an optical signal, e.g., fluorescence, therein.In this embodiment, the electrophoresis cartridge also includes a sourceelectrophoresis separation medium and, optionally (d) sources of liquidreagents, such as water and lysis buffer, delivered through outlets inthe electrophoresis cartridge to the system.

A control sub-system can include a computer 1973 programmed to operatethe system. The control sub-system can include user interface 1977 thatreceives instructions from a user which are transmitted to the computerand displays information from the computer to the user. The userinterface 1977 may be as described in U.S. Provisional PatentApplication Ser. No. 62/067,429, filed Oct. 22, 2014, which is entirelyincorporated herein by reference. Optionally, the control sub-systemincludes a communication system configured to send information to aremote server and to receive information from a remote server.

A sample preparation module includes a cartridge module assemblyconfigured to engage and operate one or more than one sample cartridge.A sample cartridge is configured to receive one or more samples and toperform nucleic acid extraction and isolation, and DNA amplificationwhen the cartridge is engaged with a cartridge module assembly in thesystem. It can also include controls and standards for assisting inanalysis.

The sample preparation module can include a receptacle for receiving oneor more cartridges, an engagement assembly to engage the cartridge; afluidic manifold configured to engage ports in a cartridge and todeliver pressure and/or fluids to the cartridge through the ports; adelivery assembly configured to deliver reagents, such as amplificationpre-mix, from a compartment in the sample cartridge to an amplificationcompartment (e.g., plungers to push ball valves into an open position; apneumatic manifold configured to engage ports in a cartridge and todeliver positive or negative pressure to the cartridge through the portsfor moving fluids and operating valves, pumps and routers in thecartridge; a pump configured to deliver pressure to the fluidic andpneumatic manifold. Consumable reagents can be carried in a module,e.g., a buffer module, that is, removably engageable with the cartridgemodule.

The system also can include an interface adapted to mate with a pressureport in the cartridge and to transmit positive or negative pressure froma pressure source to the port and into the fluidic circuit. Theinterface can include a nozzle which the port fits. It can include asyringe for applying positive or negative pressure from another source.

PCR can be carried out using a thermal cycler assembly. This assemblycan include a thermal controller, such as a Peltier device, infraredradiation source, circulating water, movement of constant temperatureblocks, or other material, which can be configured to heat and cool forthermal cycling and can be comprised in the cartridge module which canbe configured to move the thermal controller into thermal contact withthe thermal cycling chambers, for example, through a heat spreader (orthermoconductor that can spread/distribute heat and cooling) disposedover each reaction chamber. In some embodiments, the cartridge comprisesa temperature regulator assembly having one or more (e.g., a plurality)of thermocycling chambers and the sample cartridge can be in fluidcommunication with a fluidic channel.

An analysis and detection module is configured to receive analyte fromthe sample preparation module and perform capillary electrophoresis onthe analyte to detect analytes separated by electrophoresis and toanalyze the detected analytes. It can include a capillaryelectrophoresis assembly, a detection assembly, and an analysisassembly.

The capillary electrophoresis assembly can include an injectionassembly, that can include a denature heater assembly, a positioningassembly for positioning an analyte for capillary injection; a cathodeassembly; a capillary assembly; an anode assembly; a capillary fillingassembly for filling a capillary with separation medium and a powersource for applying a voltage between the anode and the cathode.

A detection assembly can comprise a laser configured to illuminate thecapillaries and a detector. The laser can be configured to excitefluorescent dyes in the analyte. In alternative embodiments, the lasercan be replaced by an alternate light source such as an LED. Thedetector can include a CCD array, CMOS array, photomultiplier, diodearray, or other detector, for detecting light produced by excited dyesand for producing an output signal.

An analysis assembly can include a computer comprising memory and aprocessor for executing code (e.g., code on a tangible medium) foranalyzing the output signal and producing a computer file containing ananalysis of the signal. Such an analysis can include, for example,identification of alleles from various STR loci. The computer file canbe in a format that is compatible with public databases. For example,the file can be in CODIS format which is compatible with the NationalDNA Index System (NDIS) operated by the FBI.

The system can be operated by a control module. The control module caninclude a user interface configured to receive instructions from anddeliver information to a user. It can include software programmed toexecute routines for performing the operations mentioned, above, andtransmit and receive information, such as computer files, from remotelocations, for example, over the internet.

FIG. 27A and FIG. 27B present the system of FIG. 19 in further detail.As described above and elsewhere herein, a sample cartridge interface1500 and an electrophoresis interface 2705 are comprised in the system,for engaging the sample cartridge and the electrophoresis cartridge.Both the sample cartridge and the electrophoresis cartridge can bereleasably or removably engaged with the system. The system of FIG. 19,FIG. 27A and FIG. 27B can be used in forensic analysis to decode thegenetic information of a single sample. In some cases, the system may beused to determine the genetic profile of a sample in less than about 6hours, 5 hours, 4 hours, 3 hours, 2.5 hours, 2 hours, 1.5 hours, 1 hour,30 minutes, 20 minutes, 10 minutes, 5 minutes 1 minute or less. Suchtime may depend upon, for example, the number of steps included insample processing operations.

A schematic of the system of FIG. 19, FIG. 27A and FIG. 27B isillustrated in FIG. 18. A chassis 2800 is included for structuralsupport, which may be formed of a metallic material, such as aluminum orsteel, a polymeric material, or a combination thereof. In some cases,the chassis may be structured to minimize the weight of the system. Auser interface which comprises system electronic controls 2801, embeddedcomputer 2802, and a user interface screen capable of identifying andreading fingerprint 2804 and sample patch barcode 2805, is included inthe system. The user interface receives and processes requests orinstructions from and delivers information to a user. It can includesoftware programmed to execute routines for performing the operationsmentioned, above, and transmit and receive information, such as computerfiles, from remote locations, e.g., over the internet. The userinterface can also enable the user to monitor the progress of theoperation and make changes to the operation of system if measurementsare not within selected parameters. A sample cartridge interface 2806 isprovided for receiving a sample cartridge for sample processing. Thesample cartridge described herein can be configured to receive one ormore samples and to perform at least one of sample isolation,extraction, purification, amplification or dilution, when the samplecartridge is engaged with the sample cartridge interface of the system.Sample amplification can include polymerase chain reaction (PCR). One ormore reagents that are needed for performing one or more steps of sampleprocessing may be pre-loaded or comprised in the sample cartridge, forexample, washing buffer, lysis buffer, diluent, amplification reagents,or internal lane standards. Also comprised in the system is a fullyintegrated electrophoresis cartridge 2807 which is releasably engageablewith the system via an electrophoresis cartridge interface. Theelectrophoresis system comprises all essential parts for performing anelectrophoretic analysis, such as an electrophoresis capillary,electrodes (e.g., anode and cathode), electrophoresis separation medium,or electrophoresis buffer. In some cases, it may comprise reagent thatcan be used to perform STR analysis. It may further comprise one or morereagent container for holding reagents that are used for sampleprocessing, e.g., a lysis buffer container. The lysis buffer may beplaced in fluidic communication with the sample cartridge and used forisolating the target material out of the sample during sampleprocessing, after both the sample cartridge and the electrophoresiscartridge are engaged with the system. Once the engagement of theelectrophoresis cartridge is completed, at least one automaticcommunication between the electrophoresis cartridge and the system maybe established, for example, an electrical communication 2813 betweenthe electrophoresis cartridge and the system electronic controls 2801,an optical communication 2814 between a portion of the electrophoresiscapillary in the electrophoresis cartridge and an optics module 2803 ofthe system, a fluidic communication 2815 between a sample inlet port ofthe electrophoresis cartridge and a sample outlet port of the samplecartridge, a mechanical and thermal 2816 communication between theelectrophoresis cartridge and a motorized drives and cooling module 2808of the system.

In one example, the integrated electrophoresis cartridge 2807 has all orsubstantially all of the components necessary for electrophoresis in acompact unit that is readily insertable into and removable from theelectrophoresis cartridge interface. This may permit a user to readilyengage the cartridge 2807 with the system without having to open thesystem. In some examples, all or substantially all of the componentsnecessary for electrophoresis (e.g., anode, cathode and at least oneelectrophoresis capillary are included on a single board or support ormultiple boards or supports that are securably integrated with oneanother.

The system provided herein may further comprise a power source 2812 forsupplying the power for the system, AC power source 2811 for applying avoltage gradient across the anode and the cathode, one or more fans 2810for dissipate the heat for one or more parts of the system, and one ormore USB ports 2809 for collecting and transferring data either withinthe system or outside the system.

Also provided herein, the electrophoresis cartridge may comprise one ormore of sub-containers or sub-cartridges that are removably insertablein the electrophoresis cartridge, such as, sub-containers for holdingelectrophoresis separation medium, reagents for sample processing, orreagents for sample analysis. FIG. 24 shows an example of anelectrophoresis cartridge comprising an electrophoresis separationmedium sub-container. As shown in FIG. 24, an electrophoresis cartridgeis manufactured to have a space 2902 configured to specifically receiveand accommodate a secondary or sub-container. A sub-container 2901 usedfor holding the electrophoresis separation medium can be stored outsidethe electrophoresis cartridge 2903 before the engagement of theelectrophoresis cartridge with the system. The sub-container which holdsthe electrophoresis separation medium may be installed 2905 into theelectrophoresis cartridge a short time before the engagement of theelectrophoresis cartridge with the system, for example, less than 1hour, 50 minutes, 40 minutes, 30 minutes, 20 minutes, 15 minutes, 10minutes, 5 minutes before engaging the electrophoresis cartridge withthe system. Once the electrophoresis cartridge is installed 2906 intothe system, the sub-container may be placed in thermal communicationwith a thermal control module of the system, which may adjust thetemperature of the sub-container to a desired value and maintain it fora period of time.

FIG. 25A and FIG. 25B show clear shell views of an exampleelectrophoresis cartridge of the present disclosure. In general, theelectrophoresis cartridge may comprise a cartridge casing 400, asub-container (or sub-cartridge) casing 401, an optical interface 402for providing a light source and detecting signals from analytes, one ormore hydrodynamic devices (e.g., fluid coupling) 403, an anodesub-assembly 404, a cathode sub-assembly 405, an electrophoresiscapillary 406, an electrical interface 407, one or more mechanicalinterfaces (e.g., 408, 409, 412 and 413) for applying pressure or forceson parts of the electrophoresis cartridge, a thermal interface 410 forcontrol the temperature of the sub-container 401, and an electricalinterface 411 for providing a voltage between at least one anode in theanode sub-assembly and at least one cathode in the cathode sub-assembly.

A schematic of an example electrophoresis cartridge 500 is shown in FIG.26. The electrophoresis cartridge 500 may comprise an electrophoresisassembly which includes an anode sub-assembly 507, a cathodesub-assembly 510, and at least one electrophoresis capillary 509 to beused in sample separation and analysis for at least one sample.

As shown in FIG. 26, three cathode nodes 511 can be included in thecathode sub-assembly and in fluidic and electrical communication with afirst end of the electrophoresis capillary. At least one anode node 529is comprised in the anode sub-assembly and in fluidic and electricalcommunication with a second end of the electrophoresis capillary.Although presented in the present disclosure are an anode sub-assemblyand a cathode sub-assembly comprising one anode node and three cathodenodes, it shall be appreciated that any positive number of anodes andcathodes may be used, dependent upon, different applications.

The electrophoresis cartridge may also comprise more than one containerfor holding electrophoresis separation medium and more than one reagentfor sample processing and analysis. As shown in FIG. 26, anelectrophoresis separation medium container 501, a first reagentcontainer 502, a second reagent container 503, a third reagent container504, and a fourth reagent container 505 are included in theelectrophoresis cartridge 500. The containers may be configured to beremovably insertable into the electrophoresis cartridge. After each runof sample analysis, one or more containers may be replaced or reused,depending upon, the applications. In some cases, it may be desirable tostore or transport one or more containers at a pre-determinedtemperature, for example, if a thermal-sensitive electrophoresisseparation medium or electrophoresis reagent is used and the trivialchange of temperature may impair its performance and thereafter resultin the degradation or failure of the whole analysis. Once the containersare installed in the electrophoresis cartridge, they may be kept at thesame or a different temperature. Installation of the containers may berealized manually or automatically.

Once the electrophoresis separation medium container 501 is properlyengaged with the electrophoresis cartridge, with the aid of a firstmechanical interface 515 for controlling one or more fluid handlingdevices (e.g., a pump), the electrophoresis separation medium may bedriven and moved into the anode sub-assembly 507. The electrophoresisseparation medium may be further pushed into at least one of theelectrophoresis capillary with the application of a second and a thirdmechanical interface 514 and 516 by exerting a force (or pressure) on ahigh pressure piston 506 and an anode main piston (not shown).

Any suitable reagent may be used in the present disclosure. Reagents maybe solid, semi-solid or liquid. In cases where liquid reagents are used,they may comprise organic fluid, inorganic fluid, or a mixture thereof.For example, reagents may comprise water, electrophoresis buffer, sampleprocessing buffer (e.g., a lysis buffer), loading buffer, regenerationfluid, or combinations thereof. For example, reagents can be provided(e.g., stored) in an aqueous solution, or can be provided (e.g., stored)in a solid or dry (e.g., lyophilized) form and then placed into solutionby addition of a liquid (e.g., an aqueous solution) as appropriate.Alternatively, reagents can be provided (e.g., stored) in asubstantially water-free non-ionic organic solvent (e.g., an alcoholsolvent) or in a substantially water-free ionic organic solvent (e.g., adeep eutectic solvent) and can be re-hydrated by addition of an aqueoussolution as appropriate, as described in PCT Patent Publication No. WO2014/055936, which is entirely incorporated herein by reference. As usedin the present disclosure, the term “regeneration fluid” generallyrefers to a fluid that is able to renew or restore the function orperformance of one or more parts of the electrophoresis cartridge, forexample, the electrophoresis capillary. In some cases, the regenerationfluid may comprise an aqueous solution. In some cases, the regenerationfluid may comprise an alkaline fluid. In some cases, the regenerationfluid may comprise one or more alkali hydroxides.

To collect any liquid or fluid from, for example, an electrophoresiscapillary, the anode sub-assembly, or the cathode sub-assembly, twowaste containers 512 and 513 may be included in the electrophoresiscartridge and communicate with the anode-subassembly and the cathodesub-assembly, respectively. Any number of waste containers (e.g., atleast 1, 2, 3, 4, or 5) may be included in the electrophoresiscartridge, as provided in the present disclosure. For example, besidesthe waste containers that are in communication with the anodesub-assembly and cathode sub-assembly, each of the reagent containersand the electrophoresis separation medium container may be provided withits water container.

In some aspects of the present disclosure, the electrophoresis cartridgemay further comprise a plurality of fluid handling devices which placevarious parts or components of the electrophoresis cartridge in fluidiccommunication. As described above and elsewhere herein, any type ofdevices that is capable of moving or transferring the fluid may be used,such as valves, pumps, electrostatic fluid accelerators, and variousother forms of process equipment. As shown in FIG. 26, pumps 520 and 521are used to drive the reagents stored in the first and the secondreagent containers 502 and 503 to the anode sub-assembly 507, throughtheir respective fluid conduits. Similarly, pumps 522 and 523 areutilized to transfer the reagents kept in the second and the thirdreagent containers 503 and 504 to the cathode sub-assembly 510, throughtwo separate fluid conduits.

In some cases, it may be desirable that at least one of the reagentcontainers communicate with more than one part of the electrophoresiscartridge or the system. For example, as illustrated in FIG. 26, thesecond and the third reagent containers 503 and 504 are placed incommunication with parts outside of the electrophoresis cartridge,besides their communication with the cathode sub-assembly 510 of theelectrophoresis cartridge as described above. In detail, both of thereagent containers are in fluidic communication with the samplecartridge interface 519 and a fluid handling device 518 through a fluidline. A four-port valve 528 and a three-port valve 527 are utilized todirect, control and regulate different types of fluid flow in the fluidline. Alternatively or additionally, it may be advantageous to have oneor more reagent containers installed inside the electrophoresiscartridge which communicate directly with parts or components which areoutside of the electrophoresis cartridge. For example, in the presentexample as shown in FIG. 26, a fourth reagent container 505 is engagedwith the electrophoresis cartridge and placed in fluidic communicationwith the sample cartridge interface 519 through a fluid line. In somecases, one or more hydrodynamic devices (e.g., fluid couplings 524, 525and 526) may be included in the electrophoresis cartridge which may aidin delivering and transferring the reagents, analytes or samples throughthe fluid line.

The electrophoresis cartridge may also comprise a sample deliveryassembly comprising at least one sample inlet port and at least onesample line, with each sample line placing a sample inlet port incommunication with the first end of the electrophoresis capillarythrough a passage in the cathode sub-assembly. The sample inlet port maybe further configured to communicate with a sample outlet port comprisedin a sample cartridge interface 519, via a hydrodynamic device 526, forexample, a fluid coupling or a hydraulic coupling. With the sampledelivery assembly, the processed sample from a sample cartridge that isengaged with the sample cartridge interface may be directed to aseparation channel (e.g., an electrophoresis capillary) via the sampleline. Any suitable method for moving the prepared sample into theseparation channel may be used in the context of the present disclosure.For example, field-amplified stacking (FAS) may be performed bypositioning in an electrophoresis sample line a diluted mixturecomprising the sample of lower salt concentration or lower ionicstrength than used in the separation gel. In another example, a bolus ofa material (e.g., air) can be positioned downstream of the sample in thesample line, wherein the material has an electrical conductivity thatdiffers from the electrical conductivity of the electrophoresis bufferor the sample. When the sample is positioned across the separationchannel, the sample can be electrokinetically injected into theseparation channel at an appropriate voltage (e.g., about 3 kV to about5 kV, or about 4 kV) over an appropriate amount of time (e.g., about 10sec to about 20 sec, or about 15 sec). In some other examples, a pumpmay be used to drive the sample into the separation channel.

Once the prepared sample is moved into the separation channel, thesample may then be subjected to sample separation and analysis withinthe separation channel, with the aid of an electric field, as can begenerated upon the application of a voltage gradient across the anode529 and the cathode 511. Upon the effect of the electric field, analytesin the electrophoresis capillary move through the matrix (i.e.,electrophoresis separation medium) at different rates, determined by oneor more factors, such as mass, charge, or a combination thereof.

A portion of the electrophoresis capillary can be used as an opticalwindow 508 which is capable of receiving a light from a light source andemitting signals that can be captured and detected by one or moredetectors included in a detection assembly. In some cases, an opticsmodule may be provided, which may comprise both the light source and thedetection assembly. The light source is positioned to deliver a beam toat least one electrophoresis capillary via the optical window. One ormore optical detectors (e.g., charge-coupled device (CCD), complementarymetal-oxide-semiconductor (CMOS), photodetector, photo diode, orphotomultiplier detector) may be optically coupled to receive signalsemitted from at least one electrophoresis capillary through the opticalwindow. As discussed elsewhere in the present disclosure, the opticalcommunication between the optical window in the electrophoresiscartridge and both the light source and the optics module may beautomatically established at the same time as the occurrence of theengagement of the electrophoresis cartridge.

The capillary can be heated to maintain an appropriate runningtemperature. The capillary can be heated by, for example, flowingtemperature-controlled air over the capillary, by placing the capillaryin thermal contact with a thermoelectric heater (e.g., a Peltier), or byplacing the capillary in thermal contact with a resistive heater. Anexample of an assembly using a resistive heater to heat a capillary isdescribed in U.S. Patent 8,512,538. In another embodiment, a capillaryheater assembly can comprise a flexible heater circuit comprising aresistive heater (e.g., a wire trace) in a polymeric substrate. Suchflexible heater circuits are commercially available from Mod-Tronic(Thermofoil®), Kapton (polyimide heater) and McMaster-Carr (ultrathinheat sheet).

VI. Method of Using

The cartridges of this disclosure can be used in an integrated systemfor preparing a sample, for example, DNA isolation and amplification.For example, in one embodiment, a sample contained on for example a swabor a card punch, can be introduced into sample chamber 120. Thecartridge can be engaged with cartridge interface 901. Cell lysis buffercontained in an off-chip reservoir can be feed through port 112 into thefluidic channel in the cartridge and into the sample chamber 120 byclosing valves 141, 142, 143, and 147. Port 112 can be connected to asyringe or to another source of positive or negative pressure. Afterlysis, lysate can be moved through a fluidic channel on the cartridge,for example, with a plunger that applied vacuum through port 112 to drawthe fluid into reaction chamber 122 by opening valves 147, 148, 149, and142; and closing valves 146, 145, 143, 144, and 141. In one embodiment,the DNA reaction chamber can include material that captures apre-determined amount of analyte. Excess fluid can be moved into wastechamber 123 while the reaction chamber is filled. Reagents forperforming PCR or other reactions can be introduced into the reactionchamber through ports 115 and 116. In one embodiment, as detailed in USPatent application US 2013/0115607 and International Patent ApplicationWO 2013/130910, an actuator pushes on ball valves, e.g., 803, to pushthe master mix in port 115 and the primers in port 116 into reactionchamber 122. A thermal control mechanism in the system can apply heat toperform thermal cycling in reaction chamber 122 of the cartridge withvalves 148 and 149 closed. Following thermal cycling, valves 148, 145,143 and 141 are opened and valves 149, 146, 147, 144, 142 are closed,and internal lane standard is dispensed from port 114 into reactionchamber 122 and pushed into mix chamber 121. Following mixing, valves141, 142, 145, 146 are closed; valves 143 and 144 are opened and theamplified STR mixture with internal lane standard is pushed to throughport 113 to a capillary electrophoresis analysis module for separation,detection, and analysis.

A. Amplification and Cycle Sequencing—One Channel

In another embodiment, cartridges of this disclosure can be used toperform DNA amplification and subsequent preparation for cyclesequencing. The target for sequencing can be, for example, a diagnostictarget, such as a gene for genotyping, a polynucleotide bearing asomatic mutation, e.g., from a tumor, or a polynucleotide from aninfectious microorganism such as a bacterium or a virus.

An exemplary cartridge 2100 for such an embodiment is shown in FIG. 21.Cartridge 2100 has input 2110. A sample 2105 can be introduced intosample chamber 2120. The cartridge can be engaged with an interface ofinstrument 2180 configured to supply reagents and motive forces. Celllysis buffer contained in an off-chip reservoir 2182 can be fed throughport 2112 into a fluidic channel in the cartridge and into the samplechamber 2120 by opening valve 2114. Port 2112 can be connected to asyringe 2170 or to another source of positive or negative pressure.

After lysis, lysate can be moved through a fluidic channel on thecartridge into isolation chamber 2130 by opening valve 2116; if requiredvacuum can be applied by syringe 2170 by opening valve 2118.Magnetically responsive particles, e.g., beads 2184, can be introducedinto the isolation chamber before or after introduction of the lysate byopening valve 2118. In another embodiment, the beads can be preloadedinto isolation chamber 2130. Polynucleotides can be captured on theparticles and immobilized by application of a magnetic force to theisolation chamber 2130 by magnetic actuator 2135. The particles can bewashed with, e.g., ethanol 2186, and the wash moved to a waste chamberon cartridge (not shown) or off-cartridge 2188.

Then the polynucleotides can be moved into a reaction chamber 2140 forPCR by opening valve 2122. Reagents for amplifying a specific nucleotidesequence can be introduced into the reaction chamber from sealedcompartments through ports 2113 and 2115 or these sealed compartmentscan contain the reagents in an integrated vial with seals by for exampleTeflon balls. These include primers, nucleotides, and hot start DNApolymerase. Primers are typically kept separate in a “primer mix” fromthe other ingredients, mixed as “master mix”. A thermal controlmechanism in the system, e.g., thermal cycler 2160, can apply heat toperform thermal cycling in reaction chamber 2140 of the cartridge.Following thermal cycling, remaining primers and nucleotidetriphosphates can be degraded by adding, for example, exonuclease I andshrimp alkaline phosphatase from a sealed compartment through port 2117.Following reaction, the exonuclease I and shrimp alkaline phosphatasecan be degraded by heating to 80 C by thermal cycler 2160.

Reagents for performing cycle sequencing can then be introduced into thereaction chamber, for example, from sealed compartments on the cartridgethrough ports 2119 and 2121. These include a sequencing primer,nucleotides, hot start DNA polymerase, and labeled dideoxynucleotides(e.g., Big Dye® terminators form Life Technologies®) for dye terminatorsequencing. Primers are typically kept separate in a “primer mix” fromthe other ingredients, mixed as “master mix”. Thermal cycling producesdideoxynucleotide-terminated polynucleotides with base specificfluorescent label.

This mixture can then be moved back into isolation chamber 2130.Magnetically responsive particles can be introduced into isolationchamber 2130 for polynucleotide capture and clean up.

Cleaned up polynucleotides can then be pushed, e.g., with air 2192through output port 2123 to a capillary electrophoresis analysis modulefor separation, detection, and analysis.

In alternative embodiments, some or all reagents are stored incompartments on the cartridge for use.

B. Amplification and Cycle Sequencing—Multi-Channel

In an alternative embodiment, a cartridge of this disclosure has afluidic circuit with a plurality of branches, each branch adapted toperform a separate biochemical reaction. For example, each of twobranches can be used to perform one of forward and reverse strand cyclesequencing on a sample. The forward strand can be prepared forsequencing in a first branch and the reverse strand can be prepared forsequencing in a second branch. Alternatively, different branches can beused to amplify different target nucleotide sequences from the samesample.

An exemplary cartridge 2200 for such an embodiment is shown in FIG. 22.Cartridge 2200 has input 2210. A sample 2205 can be introduced intosample chamber 2220. The cartridge can be engaged with an interface ofinstrument 2280 configured to supply reagents and motive forces. Celllysis buffer contained in an off-chip reservoir 2282 can be fed throughport 2212 into a fluidic channel in the cartridge and into the samplechamber 2220 by opening valve 2214. The lysis solution can be compatiblewith particles used to capture polynucleotides. Port 2212 can beconnected to a syringe or to another source of positive or negativepressure.

After lysis, lysate can be moved through a fluidic channel on thecartridge into isolation chamber 2230 by opening valve 2216.Magnetically responsive particles, e.g., beads 2284, can be introducedinto the isolation chamber before or after introduction of the lysate byopening valve 2218. Polynucleotides can be captured on the particles andimmobilized by application of a magnetic force to the isolation chamber2230 by magnetic actuator 2235. The particles can be washed with, e.g.,ethanol 2286, and the wash moved to a waste chamber or off-cartridge2288.

Then, aliquots of the polynucleotides can be moved into reactionchambers 2240 a-c for PCR by opening valves 2222 a-c. This can be done,for example, by opening and closing these valves sequentially, andmoving material into each open chamber. Reagents for amplifying aspecific nucleotide sequence can be introduced into the reaction chamberfrom sealed compartments or may be present in lyophilized form. Athermal control mechanism in the system, e.g., thermal cycler 2260, canapply heat to perform thermal cycling in reaction chamber 2240 of thecartridge.

Following thermal cycling, the products can be moved into chambers 2250a-c by opening valves 2242 a-c. Here, primers and nucleotidetriphosphates are degraded by, for example, exonuclease I and shrimpalkaline phosphatase present in lyophilized form or added from a sealedcompartment. Following reaction, the exonuclease I and shrimp alkalinephosphatase can be degraded by heating to 80 C by thermal cycler 2260.

The samples are then moved into reaction chambers 2250 a-c by openingvalves 2251 a-c for preparation for cycle sequencing. Again, reagentsfor performing cycle sequencing can be introduced into the reactionchamber, for example, from sealed compartments on the cartridge or maybe present in lyophilized form. Thermal cycling producesdideoxynucleotide-terminated polynucleotides with base specific labels.

The product of the thermal cycling reactions is then moved into clean-upchambers 2254 a-c by opening valves 2253 a-c. Magnetically responsiveparticles can be introduced into clean-up chambers 2254 a-c forpolynucleotide capture and clean up.

Cleaned up polynucleotides can then be pushed, e.g., with air 2292through output ports 2223 a-c by opening valves 2255 a-c to a capillaryelectrophoresis analysis module for separation, detection, and analysis.

C. DNA Quantification

In another embodiment, cartridges of this disclosure include a DNAquantification function. Such a function can be useful to meter anamount of DNA for amplification determined appropriate for down-streamapplications such as STR amplification.

An exemplary cartridge 2300 for such an embodiment is shown in FIG. 23.Cartridge 2300 has input 2310. A sample 2305 can be introduced intosample chamber 2320. The cartridge can be engaged with an interface ofinstrument 2380 configured to supply reagents and motive forces. Celllysis buffer contained in an off-chip reservoir 2382 can be fed throughport 2312 into a fluidic channel in the cartridge and into the samplechamber 2320 by opening valve 2314. Port 2312 can be connected to asyringe 2370 or to another source of positive or negative pressure.

After lysis, lysate can be moved through a fluidic channel on thecartridge into isolation chamber 2330 by opening valve 2316.Magnetically responsive particles, e.g., beads 2384, can be introducedinto the isolation chamber before or after introduction of the lysate byopening valve 2318. Polynucleotides can be captured on the particles andimmobilized by application of a magnetic force to the isolation chamber2330 by magnetic actuator 2335. The particles can be washed with, e.g.,ethanol 2386, and the wash moved to a waste chamber or off-cartridge2388.

Then, a predetermined amount of the particles with captured DNA can bemoved into a reaction chamber 2340 by opening valve 2322. Magneticactuator 2335 immobilizes the beads in reaction chamber 2340.Human-specific qPCR reagents, such as Quantifiler from Thermo FisherScientific™ or Plexor HY System from Promega™, are introduced into thereaction chamber from sealed compartments through ports 2319 and 2321. Athermal control mechanism in the system, e.g., thermal cycler 2360, canapply heat to perform thermal cycling in reaction chamber 2340 of thecartridge for qPCR. A detection device 2370, e.g., using illumination,determines the course of the reaction. This information is used todetermine how much DNA is captured per unit bead volume. The amount ofbeads necessary to carry the predetermined quantity of DNA needed iscalculated.

Material in reaction chamber 2340 can then be pushed, e.g., with air,2392 through output port 2323.

Next, a volume of beads from isolation chamber 2330 determined to carrythe desired amount of DNA is moved into reaction chamber 2340. Reagentsfor performing PCR can then be introduced into the reaction chamber, forexample, from sealed compartments on the cartridge through ports 2313and 2315, and the reaction thermal cycled.

Internal ladder standard 2317 can then be pushed, e.g., with air 2392through output port 2323 to a capillary electrophoresis analysis modulefor separation, detection, and analysis.

The cartridges of this disclosure can be used in an integrated systemfor analyzing a sample, for example, DNA isolation and amplificationwith real time or end point detection. For real time measurement, thesamples can be interrogated by an optical detection system whileamplifying in reaction chamber 122. The readout can be the change influorescence or by melting point. The probes can be human specific forhuman identification, forensics, or molecular diagnostic applications,or specific for pathogens for molecular diagnostic applications, or forbioagents for biodefense applications or nonspecific intercalators fordetermining the amount of DNA present. Amplification methods include,for example, thermal or isothermal amplification reactions, for example,PCR, rolling circle amplification, whole genome amplification, nucleicacid sequence-based amplification, and single strand displacementamplification, single primer isothermal linear amplification (SPIA),loop-mediated isothermal amplification, ligation-mediated rolling circleamplification and the like.

The cartridges of this disclosure can be used in an integrated systemfor analyzing a sample. The assay can detect a polypeptide (e.g.,immunoassay) or a nucleic acid (e.g., PCR or reverse transcriptasefollowed by amplification). To detect an immunoassay, after lysis of thesample and movement of the lysed sample to reaction chamber 121, ports115 and 116 can be used to add primary and secondary antibodies to thesample. The detection can be in sample chamber 121 or the sample can bemoved through port 113 to a detector.

The assay can be multiplex or single analyte. They can involve any assayto measure the presence, amount, activity, or other characteristics ofthe sample. These include assays that involve detection by fluorescence,luminescence, chemiluminescence, absorbance, reflectance, transmittance,birefringence, refractive index, colorimetric, and combinations thereof.In this instant disclosure, the enzyme master mix and the substratemight be individually added to the reaction and the progress or endpointof the assay monitored optically.

In other embodiments, cartridges of this disclosure can be used toprepare samples for additional analytical devices. Analytical methodscan include sequencing, chromatography, (e.g., gas or size exclusion)electrometry, ellipsometry, refractrometry, interferometry (e.g., backscattering interferometry), spectrometry (e.g., mass spectrometry, NMRspectrometry, Raman spectroscopy, Surface-enhanced Raman Spectroscopy),surface plasmon resonance. Sequencing methods can includehigh-throughput sequencing, pyrosequencing, sequencing-by-synthesis,single-molecule sequencing, nanopore sequencing, semiconductorsequencing, sequencing-by-ligation, sequencing-by-hybridization, RNA-Seq(Illumina), Digital Gene Expression (Helicos), Next generationsequencing, Single Molecule Sequencing by Synthesis (SMSS)(Helicos),massively-parallel sequencing, Clonal Single Molecule Array (Solexa),shotgun sequencing, Maxim-Gilbert sequencing, Sanger sequencing, primerwalking, sequencing using PacBio, SOLiD, Ion Torrent, or Nanoporeplatforms.

For STR applications, after thermal cycling, other reagents such asmolecular weight markers (size standards) can be combined with the PCRproduct. Products of the PCR can be moved off chip for analysis throughan output line connected to port 113 (sample out).

In such an embodiment, when the reaction is a short tandem repeat (STR)reaction, in many jurisdictions for casework samples, the amount ofhuman DNA must be quantified. The typical forensic process is toquantify an extracted sample using real time polymerase chain reaction(PCR) in a separate instrument before the sample is STR amplified. Inthis instant disclosure, a human specific probe is added to the STRmixture which has fluorescence outside the range used by the STR kit.The reaction chamber 122 is interrogated by a suitable wavelength oflight for the human specific probe while the STR is being PCR amplified.The human specific probe can be a quencher such as a Black HoleQuencher® or a TaqMan® probe or other chemistries well know to oneskilled in the art. As the PCR cycles increase, the fluorescence fromthe human specific probe is monitored to quantify the amount of humanDNA in the reaction. In a preferred embodiment, the number ofamplification cycles can be adjusted based upon the amount of human DNAmeasured; this can be on a cartridge-by-cartridge monitoring ifindependent thermal cyclers are in use. One advantage is that the humanspecific probe will allow the concurrent STR amplification to achieve anoptimal amplification and produce an amount of STR product that isoptimal for the kit. A second advantage is the real time monitoringconcurrent with the STR amplification allows integration of asample-to-answer system without having an additional separatequantification process. A third advantage is for low copy number sampleswhere there is barely enough sample to produce a good STR profile theintegration of the quantification with the STR amplification preventsthe aliquot typically used for quantification from causing the remainingsample to not have enough DNA for a good STR amplification.

The following examples are offered by way of illustration and not by wayof limitation.

EXAMPLES Example 1

The cartridge is a polypropylene molding with an integrated syringebarrel and sample chamber with a polyethylene heat seal over the area ofthe fluidics. There is an absorbent material in the waste chamber and asmall dot of capture material in the reaction chamber. The barrel isloaded with a quantity of lysis solution (500-1000 uL) isolated betweentwo rubber plungers. There are three reagent vessels on the chip thatseal with top and bottom Teflon balls; two for the two parts of theGlobal Filer mastermix/primer which are loaded with 7-10 uL of solutionand one containing a water/ILS solution that is used as a diluent beforetransfer to the cathode.

Process Steps

1. Load cartridge

-   -   a. User removes cartridge from packaging and load into the        instrument. Instrument senses cartridge and engages. The rams        are in the retracted state.    -   b. Initial valve coining. The rams move to the closed state.

2. Load sample.

-   -   a. The sample is recorded and loaded.

3. Lysis

-   -   a. The lysis heater is turned on.    -   b. The valves are set to the appropriate position for delivery        from the syringe to the lysis chamber.    -   c. The back rubber plunger is engaged by the pump shaft on the        instrument.    -   d. The entire contents of the syringe is delivered to the lysis        chamber.    -   e. The valves are moved to the vent position.    -   f. The syringe plunger is withdrawn and the syringe fills with        air.    -   g. The valves move to the delivery position.    -   h. The air is injected into the lysis chamber to effect mixing.

4. Transfer and capture (Pulled by syringe inlet from sample chamberinto reaction chamber.)

-   -   a. The valves are set to a state where a path is open between        the lysis chamber and the waste container that passes through        the reaction chamber.    -   b. A vacuum is pulled on the waste container.    -   c. The lysate is pulled out of the chamber, through the reaction        chamber and thus over the capture media, and into the waste        where it is absorbed by the material in the chamber.    -   d. The valves are switched to the delivery position and the        plunger is brought forward.    -   e. The pull is executed again to insure all the free lysate        material is out of the chamber, through the reaction chamber and        in the waste.

5. Mastermix/primer loading and thermocycling. (Reagents pumped intoreaction chamber.)

-   -   a. The valves are set to a state where a path is open through        the waste to the vent    -   b. The two PCR mix vials are emptied into the reaction chamber    -   c. All valves are closed.    -   d. Thermocycling begins.

6. Polymer fill, concurrent with cycling

-   -   a. Open anode input and anode output valve    -   b. Flush anode    -   c. Close anode output valve    -   d. Fill capillary    -   e. Rinse Cathode

7. Mix Sample and diluents (Sample and diluents to mix compartment.)

-   -   a. The valves are set to a state where a path is open between        the reaction chamber and the mix chamber to vent.    -   b. The diluent vial is emptied up into the mix chamber.

8. Sample delivery to cathode (Product pushed to out port.)

-   -   a. The valves are set to a state with a path from the syringe to        the mix chamber to the sample outlet.    -   b. The syringe is driven in to place the sample.

9. Sample injection and run

-   -   a. The sample is injected into the capillary    -   b. The buffer pump sweeps the sample out of the capillary and        flushes the lines    -   c. Electrophoresis and detection is run.

Example 2

Another protocol, performed on cartridge 1801, includes the followingsteps. Valve configurations are shown in FIG. 28.

-   -   1 Load Sample Cartridge    -   2 Prime Lysis to waste*    -   3 Dispense Lysis to Lysis Chamber    -   4 Mix Lysis with Air    -   5 Mix and Heat Lysis    -   6 Pull Lysate to Waste via Reaction Chamber    -   7 Push Primer Mix and Master Mix to Reaction Chamber    -   8 Thermal Cycling    -   9 Push Internal Lane Standard and Product thru Reaction Chamber        to Mix Chamber    -   10 Push Residual Internal Lane Standard and Product to Mix        Chamber with Air Pump    -   11 Push Product to Cathode    -   12 Water Rinse of Mix Chamber and Product Output to Cathode    -   13 Water Rinse of Mix Chamber and Reaction Chamber    -   14 Flush Water out of Sample Cartridge to Waste Chamber    -   15 Flush water from Sample Cartridge and Line to Cathode    -   16 Release        -   1. All 500 ul will be dispensed into the lysis chamber and            then pushed into the waste chamber after step #5.        -   2. Residual Primer Mix and Master Mix in line to Reaction            Chamber just below B0, will remain in Sample Cartridge after            the run.        -   3. Flush Sample Cartridge free of water.

Example 3

Another protocol, performed on a cartridge, includes the followingsteps. All valves begin in open configuration.

-   -   1. Close valves 143 and 148. Load Sample Cartridge.    -   2. Push lysis buffer from port 112 through valve 142 to Lysis        Chamber 120    -   3. Mix Lysis with Air: Push air through the following open        valves and forcing air through the circuit: 112→(open)        143→123→141→122→145→120    -   4. Mix and Heat Lysis    -   5. Pull Lysate to Waste via Reaction Chamber        120→145→122→123→143→112    -   6. Close valve 145; Push Primer Mix 154 and Master Mix 153 to        Reaction Chamber 122    -   7. Close valve 141; Thermal Cycling    -   8. Open valve 141; Push Internal Lane Standard 152 and Product        thru Reaction Chamber 122 to Mix Chamber 121    -   9. Push Product to Cathode 112→148→121→147→113

Example 4

This example shows a method to perform cycle sequencing on a nucleicacid. (Refer to FIG. 21.)

Raw sample

-   -   Lyse with chaotroph

Bead purify DNA

-   -   Move to magnetic bead processing chamber    -   Add paramagnetic beads    -   Wash 2× with diluted ethanol    -   Elute DNA or move beads to reaction chamber(s)

Perform PCR amplification (make enough of target region to sequence; ifmultiple regions are being sequenced, the sample had to be split orparallel samples for each loci)

-   -   Add PCR primers and premix with enzyme from vials 2113 and 2115    -   Thermal cycle

Exol/SAP (destroys PCR primers and nucleotide triphosphates)

-   -   Add Exo/SAP reagents (Exonuclease I/Shrimp Alkaline Phosphatase)    -   Incubate 37° C./for 15 min    -   Heat to 80° C. for 15 min

Cycle sequence

-   -   Add cycle sequencing primer and premix with enzyme and BigDye        terminators    -   Thermal cycle

Cleanup cycle sequencing products using paramagnetic beads

-   -   Move to magnetic bead processing chamber    -   Add beads and chaotroph    -   Wash 2× with diluted ethanol    -   Elute into water or buffer

Send products to capillary electrophoresis

Example 5

This example shows a method to perform amplification of markers, e.g.,diagnostic markers, followed by cycle sequencing of the amplificationproduct. (Refer to FIG. 22.)

Raw sample

-   -   Lyse with chaotroph (on cartridge or instrument)    -   Bead purify DNA        -   Add beads to isolation chamber        -   Wash 2× with dilute ethanol        -   Elute DNA or move beads    -   Purified DNA produced    -   Split purified DNA to reaction chambers in aliquots

Perform PCR amplification on each locus of interest in separate chambers

-   -   Add PCR primers and premix with enzyme    -   Thermal cycle

Destroy PCR primers and nucleotide triphosphases

-   -   Add Exo/SAP reagents (Exonuclease I/Shrimp Alkaline Phosphatase)    -   Incubate 37° C. for ˜15 min, heat to 80° C. for 15 min

Cycle sequence

-   -   Add cycle sequencing primer and premix with DNA polymerase and        labeled dideoxy terminators    -   Thermal cycle

Cleanup cycle sequencing products

-   -   Move cycle sequencing products to cleanup chamber and perform        bead-based cleanup        -   Add beads and chaotroph        -   Wash 2×        -   Elute

Send products to capillary electrophoresis

Example 6

This example shows a method to quantify amount of human DNA before STRamplification for human identification or diagnostic fragment sizing(Refer to FIG. 23.)

Raw sample

-   -   Lyse with lysis buffer (chaotroph or something else)—bubble+heat

Bead purify DNA

-   -   Move lysate to magnetic bead processing chamber    -   Add beads    -   Wash 2×    -   Move 10% beads to reaction chamber where the beads are captured        by another magnet

Perform PCR quantification

-   -   Add Quantifiler primers and master-mix    -   Thermal cycle    -   Excite and detect signal and determine Ct    -   Stop after reaching Ct and calculate bead dilution (optimized        for downstream STR chemistry)    -   Wash reaction chamber

STR amplification in reaction chamber

-   -   Pump beads from magnetic bead processing chamber through        reaction chamber into waste adjusting the amount of beads        captured by using the qPCR optics (with beam splitters)—i.e.        “counting” beads . Actuate magnet to capture correct dilution of        beads in reaction chamber.    -   Add STR premix and master mix    -   Thermocycle

Cleanup STR amp products using a bead-based cleanup (optional based onquantification)

-   -   Add beads and chaotrophs to reaction chamber    -   Wash 2× with dilute ethanol    -   Elute and keep beads on magnet

Add ILS

Move amp product to capillary electrophoresis system

Example 7

Analyzing a sample with an electrophoresis cartridge comprising system

The electrophoresis system is highly integrated and configured toremovably engage with a system for sample preparation, processing andanalysis. In general, the system comprises there fully-integrated andautomated components, i.e., a user interface, a sample cartridgeinterface and an electrophoresis cartridge interface. The samplecartridge interface and the electrophoresis cartridge interface areconfigured to releasably engage a sample cartridge for sample processingand an electrophoresis cartridge for sample analysis. The user interfacefurther comprises a control module, a user interface screen and anembedded computer system. The user interface is configured to read andidentify the fingerprint of a user and barcodes of sample packaging. Auser inputs one or more instructions or requests via the user interfacescreen and the embedded computer processes the requests and transformsthe requests into signals which then initiate the operation of thesystem.

A user removes an electrophoresis cartridge from packaging and load intothe instrument. Instrument senses the cartridge and engages. Multiplecommunications between the electrophoresis cartridge and the systemincluding: (i) a fluidic communication between an inlet port of theelectrophoresis cartridge and an outlet port of a sample cartridgecomprised in the system, (ii) an electrical communication betweenelectrodes (i.e., anode and cathode) of the electrophoresis cartridgeand a power source of the system, (iii) an optical communication betweenan optical window of the electrophoresis cartridge and an optics moduleof the system, (iv) a first thermal communication between theelectrophoresis capillary and a first thermo control assembly of thesystem, (v) a second thermal communication between a gel sub-cartridgeof the electrophoresis cartridge and a second thermal control assemblyof the system, (vi) a first mechanical communication between an anodesub-assembly of the electrophoresis cartridge and a first mechanicalinterface of the system, (vii) a second mechanical communication betweena cathode sub-assembly of the electrophoresis cartridge and a secondmechanical interface of the system, and (viii) a magnetic communicationbetween the electrophoresis cartridge and the system, are establishedconcurrently with the engagement of the electrophoresis cartridge.

Electrophoresis gel stored in the gel sub-cartridge is pumped andinjected into the electrophoresis capillary by a high pressure pistoncomprised in the anode sub-assembly. As the gel is injected, a washingbuffer is pumped into a passage of the cathode sub-assembly for removingexcessive gel in the cathode sub-assembly. Subsequently, a preparedanalyte is directed into the electrophoresis capillary from the sampleline in the cathode sub-assembly. A voltage gradient is then appliedacross two ends of the electrophoresis capillary to performelectrophoretic analysis and separate different components of theanalyte which emit distinguishable optical signals upon the illuminationof a laser. The signals are detected by a CCD camera comprised in theoptics module and subjected to further analysis. Conclusion is drawnbased on the results.

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

While certain embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

1. A cartridge comprising: (a) a cartridge body comprising a malleablematerial and having, disposed on a surface of the body, at least onevalve body comprising a valve inlet and a valve outlet, each fluidicallyconnected to a fluidic channel; and (b) a layer comprising a deformablematerial bonded to a surface of the cartridge body and sealing the atleast one valve body at points of attachment, thereby forming at leastone valve; wherein the at least one valve body is depressed in thecartridge body relative to the points of attachment and wherein thedeformable material covering the at least one valve body retainssufficient elasticity after deformation such that in a ground state thevalve is open.
 2. The cartridge of claim 1, wherein the malleablematerial has undergone a plastic deformation as a result of initialvalve closure.
 3. The cartridge of claim 1, wherein the at least onevalve body is a plurality of valve bodies.
 4. The cartridge of claim 1,wherein the points of attachment comprise ridges elevated above thesurface.
 5. The cartridge of claim 1, wherein the valve inlet and thevalve outlet are fluidically connected with fluidic channels.
 6. Thecartridge of claim 1, wherein the malleable material comprises aplastic, a wax or a soft metal.
 7. The cartridge of claim 1, wherein themalleable material comprises a thermoplastic, a thermoset, a singlecomponent resin or a multi-component resin.
 8. The cartridge of claim 1,wherein the malleable material comprises polypropylene, polystyrene,polyethylene, polyethylene terephthalate, polyester, polyamide, vinyl,poly(vinylchloride) (PVC), polycarbonate, polyurethane, polyvinyldienechloride, cyclic olefin polymer (COP), cyclic olefin copolymer (COC), orany combination thereof.
 9. The cartridge of claim 1, wherein thedeformable material comprises Santoprene a TPE (thermoplasticelasotomer) e.g., Santoprene, a blend of EPDM rubber and polypropylene.10. The cartridge of claim 1, wherein the deformable material has adurometer value of between 10 Shore D to 80 Shore D.
 11. The cartridgeof claim 1, wherein the deformable material comprises a heat sealmaterial.
 12. The cartridge of claim 1, wherein a portion of the layerof deformable material covering a valve seat does not comprise anelastomeric material, e.g., is not PDMS.
 13. The cartridge of claim 1,wherein the layer of deformable material has a higher yield strengththan the malleable material.
 14. The cartridge of claim 1, wherein thedeformable material is attached to the body through an adhesive.
 15. Thecartridge of claim 1, wherein the deformable material is welded to thebody.
 16. The cartridge of claim 1, wherein the deformable materialcomprises a material selected from polypropylene, polyethylene,polystyrene, cyclic olefin polymer (COP), cyclic olefin co-polymer(COC), mylar, polyacetate) and a metal.
 17. The cartridge of claim 1,comprising at least one fluidic circuit, wherein the at least onefluidic circuit comprises, as elements, at least one valve, at least onefluid inlet, at least one fluid outlet and at least one compartment,which elements are fluidically connected through fluidic channels. 18.The cartridge of claim 17, wherein the at least one compartment isselected from a reagent compartment, a sample compartment, a mixingcompartment, a reaction compartment and a waste compartment.
 19. Thecartridge of claim 17, wherein at least one fluid inlet or a fluidoutlet comprises a via through the cartridge body.
 20. The cartridge ofclaim 17, wherein at least one compartment is a sample compartmentconfigured to accept a swab.
 21. The cartridge of claim 17, wherein atleast one compartment is mixing chamber configured for bubbling of airthrough the mixing chamber.
 22. The cartridge of claim 17, wherein atleast one compartment is a reaction chamber comprising one or morethermally conductive walls and configured for thermal cycling.
 23. Thecartridge of claim 17, wherein at least one compartment is a wastecompartment.
 24. The cartridge of claim 1, wherein the body furthercomprises at least one reagent compartment comprising a reagent, whereinthe compartment comprises an openable seal that, when opened, puts thecompartment in fluidic communication through a via with a fluidicchannel on the surface.
 25. The cartridge of claim 1, wherein thedeformable layer retains sufficient elasticity to resist valve closurewhen negative pressure (e.g., suction) up to about 10 psi is exerted ona fluidic channel communicating with the valve.
 26. The cartridge ofclaim 1, wherein the valve body and a ram configured to close the valvehave shapes such that closure of the valve produces a pressure that isnormal to the surface of the valve body across all areas of the valvecross section, e.g., when the pressure of the ram is unidirectional. 27.The cartridge of claim 1, wherein the valve body is wider than thechannel to which it is connected.
 28. The cartridge of claim 1, whereinthe body further comprises one or more reagent compartments comprisingreagents including nucleic acid primers, nucleotides and DNA polymerasessufficient to perform PCR.
 29. The cartridge of claim 27, wherein thereagents are sufficient for performing multiplex PCR on STR loci.
 30. Anarticle comprising: (a) a cartridge body comprising a malleable materialand having, disposed on a surface of the body, at least one valve bodycomprising a valve inlet and a valve outlet, each fluidically connectedto a fluidic channel, wherein the at least one valve body comprisessloped or curved walls. 31.-48. (canceled)
 49. A method of making anarticle comprising: (a) providing a cartridge body comprising amalleable material and having, disposed on a surface of the body, atleast one valve body comprising a valve inlet and a valve outlet, eachfluidically connected to a fluidic channel; and (b) providing a layercomprising a deformable material; (c) bonding the layer to the surfaceto seal the at least one valve body at points of attachment, therebyforming at least one valve, wherein the at least one valve body isdepressed in the cartridge body relative to the points of attachment;and (d) deforming the deformable layer covering the at least one valvebody wherein the deformable layer undergoes plastic deformation andretains sufficient elasticity such that in a ground state the valve isopen. 50.-59. (canceled)
 60. An instrument comprising a cartridgeinterface and a cartridge engaged with the cartridge interface, wherein:(I) the cartridge is a cartridge of claim 1; (II) the cartridgeinterface comprises: (A) at least one mechanical actuator, eachmechanical actuator positioned to actuate a valve; (B) at least onemotor operatively coupled to actuate a mechanical actuator toward oraway from a valve.
 61. The instrument of claim 60, wherein: (I) thecartridge comprises at least one fluidic circuit, wherein the at leastone fluidic circuit comprises, as elements, at least one valve, at leastone fluid inlet, at least one fluid outlet and at least one compartment,which elements are fluidically connected through fluidic channels; and(II) the cartridge interface comprises a first port engaged with a fluidinlet and a second port engaged with a fluid outlet, and furthercomprises a pressure source configured to apply positive or negativepressure through either port to the fluidic circuit. 62.-64. (canceled)65. A method of controlling fluid flow in a fluid channel of fluidiccartridge comprising: (A) providing an instrument of claim 61, whereinat least one of the fluidic channels comprises a liquid; (B) closing avalve by actuating a mechanical actuator against the valve to force thedeformable layer against walls of the valve body; (C) releasing thevalve by retracting the mechanical actuator away from the valve body;and (D) moving the liquid through the valve by applying positive ornegative pressure to liquid in a fluidic channel.